Small molecule therapeutic compounds that reduce the incidence of intracerebral hemorrhage and brain microhemmorhages

ABSTRACT

The described invention relates to small molecule therapeutic compounds capable of reducing the incidence of intracerebral hemorrhage and brain microhemorrhages identified using zebrafish and mouse models of intracerebral hemorrhage and brain microhemorrhages.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending application number15/667,423 (filed Aug. 2, 2017), which claims the benefit of priority toU.S. provisional application No. 62/370,077 (filed Aug. 2, 2016),entitled SMALL MOLECULE THERAPEUTIC COMPOUNDS THAT REDUCE THE INCIDENCEOF INTRACEREBRAL HEMORRHAGE AND BRAIN MICROHEMORRHAGES. Each of theseapplications is incorporated by reference herein in its entirety

FIELD OF THE INVENTION

The described invention relates to small molecule therapeutic compoundscapable of reducing the incidence of intracerebral hemorrhage and brainmicrohemorrhages.

BACKGROUND

Many pathologic conditions cause a destabilization of the vascularnetwork resulting in endothelial hyperpermeability, excessive vascularsprouting, and angiogenesis. (Smith VICP, Li, DY, Whitehead, KJ (2010)“Mechanisms of vascular stability and the relationship to humandisease,” Curr. Opin. Hematol. 17(30: 237-44).

Basal Vascular permeability (BVP)

Vascular permeability is an extremely complex process that, in differentsettings, involves distinctly different types of blood vessels and makesuse of different anatomic and molecular pathways.

While the vascular system of higher organisms is often described as“closed”, it needs to be sufficiently “open” (i.e., “permeable”) toallow the ready exchange of small molecules (gases, nutrients, wasteproducts) with the tissues. (Nagy, J. A., Benjamin, L., Zeng, H ,Dvorak, A. M., Dvorak, H. F. (2008) “Vascular permeability, vascularhyperpermeability and angiogenesis,” Angiogenesis 11(2): 109-119).Plasma proteins also need to cross the normal vascular barrier, at leastin small amounts.

Permeability, meaning the net amount of a solute, typically amacromolecule, that has crossed a vascular bed and accumulated in theinterstitium in response to a vascular permeabilizing agent or at a siteof pathological angiogenesis, is an extremely complicated process thatis affected by many different variables. Id. These include the intrinsicproperties of the different types of microvessels involved (capillaries,venules, mother vessels (MV)); the size, shape, and charge ofextravasating molecules; the anatomic pathways molecules take incrossing the endothelial cell barrier; the time course over whichpermeability is measured; and the animals and vascular beds that arebeing investigated. Id.

Basal Vascular Permeability (BVP)

Molecular exchange in normal tissues takes place primarily incapillaries, largely by diffusion. The molecules exchanged consistlargely of gases (O₂ and CO₂), water, small molecules such as salts andsugars, and only small amounts of plasma proteins. Id. The extent of BVPvaries considerably in different normal tissues and is subject tosubstantial change in response to changes in hydrostatic pressure,opening of closed vessels, surface area available for exchange, bloodflow, etc. Id.

Water and lipophilic solutes (e.g., gases such as O₂ and CO₂) are ableto diffuse through endothelial cells; they also pass readily throughinter-endothelial cell junctions and through endothelial fenestrae. Id.Small lipophilic molecules can also dissolve in endothelial cellmembranes and so pass from the vascular lumen to the interstitium. Id.Capillary endothelial cells contain large numbers of small (about 70 nmdiameter) vesicles (caveolae) the majority of which are found connectedto the luminal and abluminal plasma membranes by means of stomata thatare generally closed by thin diaphragms containing plasmalemmal vesicleassociated protein (PV-1), an endothelial-specific integral membraneglycoprotein associated with the stomatal diaphragms of vaveolae,transendothelial channels, and vesiculo-vacuolar organelles and thediaphragms of endothelial fenestrae. (Stan, R V, Tkachenko, E., Niesman,I R, (2004), “PV1 is a key structural component for the formation of thestomatal and fenestral diaphragms,” Mol. Biol. Cell 15(8): 3615-30).Others are in the cytoplasm.

Acute Vascular Hyperpermeability (AVH)

A rapid increase in vascular permeability occurs when themicrovasculature is exposed acutely to any of a number of vascularpermeabilizing factors, for example, VEGF-A, histamine, serotonin, PAF,etc. Some of these agents (e.g., histamine, serotonin, VEGF-A) arenormally stored in tissue mast cells (Nagy, J. A., Benjamin, L., Zeng,H., Dvorak, A. M., Dvorak, H. F. (2008) “Vascular permeability, vascularhyperpermeability and angiogenesis,” Angiogenesis 11(2): 109-119, citingBoesiger J, Tsai M, Maurer M et al (1998) Mast cells can secretevascular permeability factor/vascular endothelial cell growth factor andexhibit enhanced release after immunoglobulin E-dependent upregulationof fc epsilon receptor I expression. J Exp Med 188:1135-1145; Galli S J(2000) Mast cells and basophils. Curr Opin Hematol 7:32-39; Galli S J(1997) The Paul Kallos Memorial Lecture. The mast cell: a versatileeffector cell for a challenging world. Int Arch Allergy Immunol113:14-22) and so may be released by agents that cause mast celldegranulation, e.g., allergy, insect bites, etc. Single exposure to anyof these permeability factors results in a rapid but self-limited(complete by 20-30 min) influx of plasma into the tissues.

The quantity of extravasated fluid in AVH is greatly increased abovethat found in BVP and its composition is greatly changed. The fluid thatextravasates in AVH is rich in plasma proteins, approaching the levelsfound in plasma, and is referred to as an exudate. Id. Among the plasmaproteins that extravasate are fibrinogen and various members of theblood clotting cascade. Id. When these come into contact with tissuefactor, a protein that is normally expressed by many interstitial cells,the clotting system is activated and the exudate clots to deposit fibrin(Id. Citing Dvorak H F, Quay S C, Orenstein N S et al (1981) Tumorshedding and coagulation. Science 212:923-924; VanDeWater L, Tracy P B,Aronson D et al (1985) Tumor cell generation of thrombin via functionalprothrombinase assembly. Cancer Res 45:5521-5525). Fibrin forms a gelthat traps water and other solutes, restraining their clearance bylymphatics or capillaries and resulting in tissue swelling (edema). Id.As long as the permeability stimulus is not continuous, the depositedfibrin is rapidly degraded without further consequences. Id.

AVH also differs from BVP in that the vascular leakage takes place frompost-capillary venules, highly specific vessels just downstream ofcapillaries (Id. Citing Majno G, Palade G E, Schoefl G I (1961) Studieson inflammation. II. The site of action of histamine and serotonin alongthe vascular tree: a topographic study. J Biophys Biochem Cytol11:607-626; Majno G, Shea S M, Leventhal M (1969) Endothelialcontraction induced by histamine-type mediators: an electron microscopicstudy. J Cell Biol 42:647-672). It has been proposed that histamine andother vascular permeabilizing agents induce endothelial cells tocontract and pull apart to form intercellular (paracellular) gaps ofsufficient size to permit plasma-protein extravasation. Id. In addition,venular epithelium contains a structure, the vesiculo-vacuolar organelle(VVO), that offers an alternative, trans-endothelial cell route forplasma extravasation in response to permeability factors (Id. citingKohn S, Nagy J A, Dvorak H F et al (1992) Pathways of macromoleculartracer transport across venules and small veins. Structural basis forthe hyperpermeability of tumor blood vessels. Lab Invest 67:596-607;Dvorak A M, Kohn S, Morgan E S et al (1996) The vesiculo-vacuolarorganelle (VVO): a distinct endothelial cell structure that provides atranscellular pathway for macromolecular extravasation. J Leukoc Biol59:100-115; Feng D, Nagy J, Dvorak A et al (2000) Different pathways ofmacromolecule extravasation from hyperpermeable tumor vessels.Microvascular Research 59:24-37; Feng D, Nagy J A, Hipp J et al (1996)Vesiculo-vacuolar organelles and the regulation of venule permeabilityto macromolecules by vascular permeability factor, histamine, andserotonin. J Exp Med 183:1981-1986; Feng D, Nagy J A, Hipp J et al(1997) Reinterpretation of endothelial cell gaps induced by vasoactivemediators in guinea-pig, mouse and rat: many are transcellular pores. JPhysiol 504(Pt 3):747-761). VVOs, which are grape-like clusterscomprised of hundreds of uncoated, cytoplasmic vesicles and vacuolesthat together form an organelle that traverses venular endothelialcytoplasm, often extend to inter-endothelial cell interfaces and theirindividual vesicles (unlike caveolae) commonly open to theinter-endothelial cell cleft. Id. The vesicles and vacuoles comprisingVVOs vary in size from those the size of caveolae to vacuoles withvolumes as much as 10-fold larger (Feng D, Nagy J A, Pyne K et al (1999)Pathways of macromolecular extravasation across microvascularendothelium in response to VPF/VEGF and other vasoactive mediators.Microcirculation 6:23-44). These vesicles and vacuoles are linked toeach other and to the luminal and abluminal plasma membranes by stomatathat are normally closed by thin diaphragms that appear similar to thosefound in caveolae. Id. It has been proposed that vascular permeabilityinducing agents cause the diaphragms interconnecting vesicles andvacuoles to open, thereby providing a transcellular pathway for plasmaand plasma-protein extravasation. Id.

Chronic Vascular Hyperpermeability (CVH)

Chronic exposure to permeability factors results in profound changes invenular structure and function that lead to the chronichyperpermeability of pathological angiogenesis as found in tumors,healing wounds, and chronic inflammatory diseases such as rheumatoidarthritis, psoriasis, cellular immunity, etc. (Id. Citing Dvorak H F(2003) Rous-Whipple award lecture. How tumors make bad blood vessels andstroma. Am J Pathol 162:1747-1757; Nagy J A, Masse E M, Herzberg K T etal (1995) Pathogenesis of ascites tumor growth: vascular permeabilityfactor, vascular hyperpermeability, and ascites fluid accumulation.Cancer Res 55:360-368). As in AVH, the fluid that extravasates is anexudate that approaches the overall composition of plasma. In tumorsfluid accumulation is generally associated with increased interstitialpressure (Id. Citing Jain R K (1988) Determinants of tumor blood flow: areview. Cancer Res 48:2641-2658); this increased pressure results frompersistent vascular hyperpermeability, clotting of the exudate withdeposition of a fluid-trapping fibrin gel, inadequate lymphaticdrainage, and the restraints imposed by surrounding tissues thattogether limit fluid dissipation. Id. These restraints are nearly absentwhen tumors grow in or around body cavities such as the peritoneum wheremassive amounts of ascites fluid can accumulate. Id.

In contrast to BVP and AVH, fluid leakage in CVH does not take placefrom any type of normal blood vessel. Instead, whether in tumors orwounds, the blood vessels that leak are newly formed, angiogenic bloodvessels; these are primarily mother vessels (MV), and also, to a lesserextent, glomeruloid microvascular proliferations (GMP) that form from MV(Id. Citing Nagy J A, Feng D, Vasile E et al (2006) Permeabilityproperties of tumor surrogate blood vessels induced by VEGF-A. LabInvest 86:767-780; Pettersson A, Nagy J A, Brown L F et al (2000)Heterogeneity of the angiogenic response induced in different normaladult tissues by vascular permeability factor/vascular endothelialgrowth factor. Lab Invest 80:99-115; Sundberg C, Nagy J A, Brown L F etal (2001) Glomeruloid microvascular proliferation follows adenoviralvascular permeability factor/vascular endothelial growth factor-164 genedelivery. Am J Pathol 158:1145-1160; Brown L F, Detmar M, Claffey K etal (1997) Vascular permeability factor/vascular endothelial growthfactor: a multifunctional angiogenic cytokine. Exs 79:233-269; Brown LF, Yeo K T, Berse B et al (1992) Expression of vascular permeabilityfactor (vascular endothelial growth factor) by epidermal keratinocytesduring wound healing. J Exp Med 176:1375-1379; Ren G, Michael L H,Entman M L et al (2002) Morphological characteristics of themicrovasculature in healing myocardial infarcts. J Histochem Cytochem50:71-79. Mother Vessels are greatly enlarged sinusoids that arise frompreexisting normal venules by a process that involves pericytedetachment, vascular basal lamina degradation, and a 4-5-fold increasein lumen size that is accompanied by extensive endothelial cellthinning. Id. Notwithstanding that Poiseuille's law indicates that bloodflow (flow rate) is proportional to the fourth power of the vascularradius, MV exhibit sluggish blood flow because of theirhyperpermeability to plasma which results in a striking increase inhematocrit. Id. The protein-rich exudates in CVH interact with tissuefactor to trigger the clotting system and deposit fibrin (Id. CitingDvorak H F, Quay S C, Orenstein N S et al (1981) Tumor shedding andcoagulation. Science 212:923-924; VanDeWater L, Tracy P B, Aronson D etal (1985) Tumor cell generation of thrombin via functionalprothrombinase assembly. Cancer Res 45:5521-5525).

Tissue factor is expressed on many tumor cells as well as hostinterstitial cells and is induced in endothelial cells by VEGF-A (Id).In addition to its fluid trapping properties, fibrin also has a numberof other properties when it persists over time as in tumors and healingwounds. It provides a pro-angiogenic provisional stroma that induces andis later replaced by the ingrowth of new blood vessels and fibroblastsand the laying down of mature fibro-vascular stroma (Id. Citing Dvorak HF (2003) Rous-Whipple award lecture. How tumors make bad blood vesselsand stroma. Am J Pathol 162:1747-1757; Dvorak H F, Orenstein N S,Carvalho A C et al (1979) Induction of a fibrin-gel investment: an earlyevent in line 10 hepatocarcinoma growth mediated by tumor-secretedproducts. J Immunol 122:166-174; Dvorak H F, Dvorak A M, Manseau E J etal (1979) Fibrin gel investment associated with line 1 and line 10 solidtumor growth, angiogenesis, and fibroplasia in guinea pigs. Role ofcellular immunity, myofibroblasts, microvascular damage, and infarctionin line 1 tumor regression. J Natl Cancer Inst 62:1459-1472). Fibrininteracts with integrins expressed by multiple cell types, therebysupporting the migration of tumor cells as well as host mesenchymalcells (endothelial cells, pericytes, fibroblasts) and inflammatory cells(neutrophils, monocytes). Id. Fibrin also sequesters growth factors,protecting them from degradation, and induces the expression ofproangiogenic molecules such as IL-8 and tissue factor. Id. Fragment E,a fibrin breakdown product, is directly pro-angiogenic (Id.).Macromolecules extravasate from MV and GMP largely via a transcellularroute (Id. Citing Nagy J A, Feng D, Vasile E et al (2006) Permeabilityproperties of tumor surrogate blood vessels induced by VEGF-A. LabInvest 86:767-780).

In short, while agents such as VEGF-A have long been known to induce AVHand CVH, apart from hemodynamic factors, much less is known about themolecular events that are responsible for the normal permeability ofBVP, and even less is known about the molecules that are involved inregulating permeability, and the molecular mechanisms that govern eachof the different types of permeability may well be different. Thesignaling pathways by which even such well-studied molecules as eNOS andcaveolin-1 act to induce permeability are poorly understood. Id. Littleis known about the molecular mechanisms that regulate such criticalevents as caveolar shuttling, the opening of VVO diaphragms, theformation of fenestrae, changes in endothelial cell junctions, etc. (Id.Citing Dejana E (2004) Endothelial cell-cell junctions: happy together.Nat Rev Mol Cell Biol 5:261-270; Oh P, Borgstrom P, Witkiewicz H et al(2007) Live dynamic imaging of caveolae pumping targeted antibodyrapidly and specifically across endothelium in the lung. Nat Biotechnol25:327-337; Ioannidou S, Deinhardt K, Miotla J et al (2006) An in vitroassay reveals a role for the diaphragm protein PV-1 in endothelialfenestra morphogenesis. Proc Natl Acad Sci USA 103:16770-16775).

Angiogenesis

Angiogenesis is a process of neovascular formation from pre-existingblood vessels during embryogenesis, adult tissue homeostasis andcarcinogenesis. (Katoh, M., (2013) “Therapeutics targeting angiogenesis:genetics and epigenetics, extracellular miRNAs and signaling networks,”Intl J. Mol. Med. 32(4): 763-67, citing Carmeliet P. (2005) Angiogenesisin life, disease and medicine. Nature. 438:932-936; Ferrara N, Kerbel RS. (2005) Angiogenesis as a therapeutic target. Nature. 438:967-974;Folkman J. (2007) Angiogenesis: an organizing principle for drugdiscovery? Nat Rev Drug Discov. 6:273-286; Carmeliet P, Jain R K. (2011)Molecular mechanisms and clinical applications of angiogenesis. Nature.473:298-307). It is distinct from vasculogenesis which is thedevelopmental in situ differentiation and growth of blood vessels frommesodermal derived hemangioblasts.

Angiogenesis occurs in multiple steps as follows: i) vasculardestabilization induced by degradation of the basement membrane anddecreased adhesion of endothelial cells; ii) angiogenic sproutingresulting from the migration of endothelial tip cells and theproliferation of endothelial stalk cells; iii) lumen formation byendothelial cells and the recruitment of pericytes to the surroundingregion of the endothelial lumen; iv) vascular stabilization depending ontight junctions and basement membrane. (Katoh, M., (2013) “Therapeuticstargeting angiogenesis—genetics and epigenetics, extracellular miRNAsand signaling networks,” Intl J. Mol. Med. 32(4): 763-67, citingCarmeliet P. (2005) Angiogenesis in life, disease and medicine. Nature.438:932-936).

Vascular endothelial growth factor (VEGF), fibroblast growth factor(FGF2), angiopoietins (ANGPT1 and ANGPT2), Notch ligands [jagged 1(JAG1) and Delta like ligand 4 (DLL4)] and transforming growth factor-β(TGF-β) regulate angiogenesis through their receptors on vascularendothelial cells. VEGF activates the endothelial nitric acid oxidesynthase (eNOS), SRC, RAS-ERK and PI3K-AKT signaling cascades throughVEGFR2 receptor on endothelial cells, which induce vascularpermeability, endothelial migration, proliferation and survival,respectively (Id. Citing Coultas L, Chawengsaksophak K, Rossant J.(2005) “Endothelial cells and VEGF in vascular development.” Nature.438:937-945; Olsson A K, Dimberg A, Kreuger J, Claesson-Welsh L. (2006)“VEGF receptor signaling-in control of vascular function.” Nat Rev MolCell Biol. 7:359-371). FGF2 promotes angiogenesis directly through FGFR1receptor on endothelial cells via signaling cascades similar to VEGF, orindirectly through VEGF secretion from endothelial cells, cardiomyocytesand stromal cells (Id. Citing Presta M, Dell'Era P, Mitola S, et al.(2005) “Fibroblast growth factor/fibroblast growth factor receptorsystem in angiogenesis.” Cytokine Growth Factor Rev. 16:159-178).ANGPT1, secreted from pericytes, activates TEK/TIE2 receptor to maintainendothelial quiescence or stabilization, whereas ANGPT2, secreted fromendothelial cells themselves by VEGF or hypoxia signaling, inhibits TEKto promote endothelial activation or sprouting (Id. Citing Fagiani E,Christofori G. (2013) “Angiopoietins in angiogenesis.” Cancer Lett.328:18-26). JAG1-Notch signaling promotes angiogenic sprouting, whereasDLL4-Notch signaling inhibits angiogenic sprouting (Id. Citing BridgesE, Oon C E, Harris A. (2011) “Notch regulation of tumor angiogenesis.”Future Oncol. 7:569-588). TGF-β signaling through TGFBR1/ALK5 receptorto the Smad2/3 cascade inhibits endothelial cell activation, maintainingendothelial quiescence, whereas TGF-β signaling through the ACVRL1/ALK1receptor to the Smad1/5 cascade promotes the migration and proliferationof endothelial cells (Id. Citing Gaengel K, Genové G, Armulik A,Betsholtz C. (2009) “Endothelial-mural cell signaling in vasculardevelopment and angiogenesis.” Arterioscler Thromb Vasc Biol.29:630-638). The VEGF, FGF, Notch and TGF-β signaling cascades aredirectly involved in the angiogenic signaling of endothelial cells (Id).

The VEGF, FGF, Notch and TGF-β signaling cascades cross-talk with WNTand Hedgehog signaling cascades to constitute the stem-cell signalingnetwork (Id. Citing Katoh M, Katoh M. (2007) “WNT signaling pathway andstem cell signaling network.” Clin Cancer Res. 13:4042-4045; Katoh Y,Katoh M. (2008) “Hedgehog signaling, epithelial-to-mesenchymaltransition and miRNA,” Int J Mol Med. 22:271-275). DVL2-bindingdeubiquitinase FAM105B regulates WNT signaling and angiogenesis (Id.Citing Rivkin E, Almeida S M, Ceccarelli D F, et al. (2013) “The linearubiquitin-specific deubiquitinase gumby regulates angiogenesis.” Nature498:318-324), while Hedgehog signaling is involved in the regulation ofliver sinusoidal endothelial cells (Id. Citing Diehl A M. (2012)“Neighborhood watch orchestrates liver regeneration.” Nat Med.18:497-499). FGF, Notch and canonical WNT signaling are involved incell-fate determination based on mutual transcriptional regulation,whereas FGF, Notch, TGF-β, Hedgehog and non-canonical WNT signaling areinvolved in epithelial-to-mesenchymal transition (EMT) due to theupregulation of SNAI1 (Snail), SNAI2 (Slug), ZEB1, ZEB2 and TWIST(Katoh, M., Nakagama, H., (2014) “FGF receptors: cancer biology andtherapeutics,” Med. Res. Rev. 34(2): 280-300). EMT is a cellular processsimilar to endothelial-to-mesenchymal transition (EndMT). Hypoxiainduces angiogenesis as a result of VEGF upregulation (Dewhirst M W, CaoY, Moeller B. (2008) “Cycling hypoxia and free radicals regulateangiogenesis and radiotherapy response.” Nat Rev Cancer. 8:425-437).Angiogenesis is orchestrated by the VEGF, FGF, Notch, TGF-β, Hedgehogand WNT signaling cascades, which directly or indirectly regulate thequiescence, migration and proliferation of endothelial cells.

During the earliest stages of angiogenesis, such as in response to theangiogenic cytokine VEGF induced by wounding and ischemia, vascularbasement membrane is degraded (Senger, D R, and David, G E, (2011)“Angiogenesis,” Cold Spring Harb. Perspect. Biol. August 3(8): a005090citing Sundberg, C. et al. (2001) “Glomeruloid microvascularproliferation follows adenoviral vascular permeability factor/VEGF-164gene delivery,” Am J Pathol 158: 1145-1160; Rowe, R G and Weiss, S J(2008) “Breaching the basement membrane: Who, when and how?” Trends CellBiol 18: 560-574; Chang S H et al. (2009) “VEGF-A induces angiogenesisby perturbing the cathepsin-cysteine protease inhibitor balance invenules, causing basement membrane degradation and mother vesselformation,” Cancer Res 69: 4537-4544). Following disruption of basementmembrane, and with the ensuing stage known as vascular sprouting (Id.Citing Nicosia, R F and Madri, J A (1987) “The microvascularextracellular matrix. Developmental changes during angiogenesis in theaortic ring-plasma clot model.” Am J Pathol 128: 78-90)), vessels becomeleaky and hyperpermeable to blood plasma proteins (Id. Citing Sundberg,C. et al. (2001) “Glomeruloid microvascular proliferation followsadenoviral vascular permeability factor/vascular endothelial growthfactor-164 gene delivery.” Am J Pathol 158: 1145-1160). This vascularhyperpermeability causes leakage of the plasma proteins fibrinogen,vitronectin, and fibronectin from the blood (Id. Citing Senger, D R(1996) “Cell migration promoted by a potent GRGDS-containingthrombin-cleavage fragment of osteopontin.” Biochim Biophys Acta 1314:13-24; Sundberg, C. et al. (2001) “Glomeruloid microvascularproliferation follows adenoviral vascular permeability factor/vascularendothelial growth factor-164 gene delivery.” Am J Pathol 158:1145-1160). Fibrinogen is subsequently converted to fibrin throughenzymatic coagulation, and together with extravasated vitronectin andfibronectin instantly transform the interstitial collagen matrix to forma new, provisional ECM. Thus, the early stages of sprouting angiogenesisare generally believed to proceed in an environment rich in preexistinginterstitial collagens in combination with fibrin, vitronectin, andfibronectin derived from the blood plasma. As vascular morphogenesisproceeds and vascular sprouts acquire lumens and mature, neovessels areagain enshrouded in vascular basement membrane with associated pericytesand thereby achieve stability (Id. Citing Grant, D S and Kleinman, H K(1997) “Regulation of capillary formation by laminin and othercomponents of the extracellular matrix.” EXS 79: 317-333; Benjamin, L Eet al. (1999) “Selective ablation of immature blood vessels inestablished human tumors follows vascular endothelial growth factorwithdrawal.” J Clin Invest 103: 159-165). Pericyte recruitment tovascular tubes directly controls this basement membrane assembly step invitro and in vivo (Id. Citing Stratman, A N et al. (2009) “Pericyterecruitment during vasculogenic tube assembly stimulates endothelialbasement membrane matrix formation.” Blood 114: 5091-5101; Stratman, A Net al. (2010) “Endothelial-derived PDGF-BB and HB-EGF coordinatelyregulate pericyte recruitment during vasculogenic tube assembly andstabilization.” Blood 116: 4720-4730). Thus, in response to stimulationwith angiogenic cytokines, angiogenesis in the adult is generallybelieved to proceed through the following basic stages: (1) degradationof vascular basement membrane and activation of quiescent endothelialcells (ECs); (2) sprouting and proliferation of ECs within provisionalECM; (3) lumen formation within the vascular sprouts, thereby creatingvascular tubes; and (4) coverage of vascular tubes with mature vascularbasement membrane in association with supporting pericytes.

Neovascularization

While often considered synonymous with angiogenesis (formation of newvessels from existing vessels), neovascularization involves a muchbroader series of temporally controlled vascular processes beginningwith angiogenesis and progressing through multiple phases resulting inthe formation of a new functional circulatory network (LeBlanc, A J etal, (2012) “Microvascular repair—post-angiogenesis vascular dynamics,”Microcirculation 19(8): 10.1111/j.1549-8719.2012.00207.x). At the onsetof neovascularization, relevant microvessel segments relax their stablevessel structure and initiate vessel sprouting leading to the formationof new vessel segments. (Id). Subsequently, the newly formed neovesselsremodel via vascular cell differentiation and incorporation ofperivascular cells into the newly formed vessel walls resulting in theappropriate density and distribution of arterioles, venules, andcapillaries. (Id). Finally, the newly formed vascular network maturesand remodels into a more efficient perfusion circuit that meets tissueperfusion needs and function. (Id).

Effective adult tissue neovascularization, whether by native ortherapeutic means, results in an expanded vascular network and increasedblood perfusion pathway length resulting in the appropriate delivery ofmore blood to tissues (Id).

While there is no one stereotypical vascular architecture, microvascularnetworks generally involve a branched network of progressively smallercaliber small arteries/arterioles at the inflow side delivering blood tothe distal capillaries which subsequently drain into a branched networkof increasingly larger caliber outflow venules/small veins, althoughthere are variations of this basic network organization, oftenreflecting tissue and/or organ specific function (Id). Each of the threegeneral vascular compartments (arterioles, capillaries, and venules)performs different functions in the microcirculation due to theirstructural and functional characteristics and their locations within thevasculature (Id). Arterioles provide the greatest resistance to bloodflow in the vascular circuit with most of this resistance attributed to1st and 2nd branch order arterioles (Id. Citing Mayrovitz H N, WiedemanM P, Noordergraaf A. (1975) “Microvascular hemodynamic variationsaccompanying microvessel dimensional changes.” Microvasc Res. 10:322-29Box 1). This is primarily due to the relative larger diameterdifferences between the feeding arteries and the smaller arterioles andthe relative fewer numbers of these proximal arterioles. The moreprevalent downstream and terminal arterioles act to broadly distributeblood throughout the tissue and control, via vessel tone dynamics, bloodflow into the most distal capillaries. The very small diameters andlarge numbers of capillaries make them ideal for supporting effectiveblood-tissue exchange of oxygen and other blood nutrients and molecules.Finally, venules, due in part to a relatively more compliant wall, serveas a high capacitance drainage system. Importantly, in a competentmicrocirculatory bed, as vessel diameters reduce within a vascularcompartment the number of vessels in that compartment increase due tobranching. This results in a sufficiently large enough cross-sectionalarea to keep resistance to blood flow across the compartment relativelylow even though resistance within a single vessel segment might be high(due to the inverse relationship between resistance and the 4th power ofthe radius). Thus, to maintain proper resistances across themicrovasculature, and therefore effective perfusion, proper branchordering is critical. In addition, blood flow distribution in a tissuedepends on the extent of branching in a logarithmic fashion (Mayrovitz HN, Tuma R F, Wiedeman M P. (1977) “Relationship between microvascularblood velocity and pressure distribution.” Am J Physiol.; 232:H400-5).This normalized relationship between vessel caliber and vessel numbers(i.e. branching) is a critical feature of functional microvascularnetwork architectures. Mismatches in this relationship lead to poorhemodynamic function typically observed as hypo-perfusion and/or hypoxiawithin the tissue. Deficits in blood perfusion (e.g. ischemia, hypoxia)are a cause of and/or complication associated with a number of diseasestates including tissue infarction, necrosis, wound healing, tissuegrafting, and organ dysfunction.

Microvascular Stability: Rho GTPase Cdc42 has been Implicated in theMediation of Endothelial Barrier Function

Endothelial adherens junctions (AJs) consist of trans-oligomers ofmembrane spanning vascular endothelial (VE)-cadherin proteins, whichbind β-catenin through their cytoplasmic domain (Broman, M T et al(2006) “Cdc42 regulates adherens junction stability and endothelialpermeability by inducing alpha-catenin interaction with the vascularendothelial cadherin complex,” Cir. Res. 98: 73-80). β-catenin in turnbinds α-catenin and connects the AJ complex with the actin cytoskeleton(Id). Rho GTPase Cdc42 regulates AJ permeability by controlling thebinding of α-catenin with β-catenin and the consequent interaction ofthe VE-cadherin/catenin complex with the actin cytoskeleton (Id).β-catenin and the associated α-catenin may then serve as support sitesfor actin polymerization, leading to formation of long endothelialplasma membrane protrusions (Kouklis, P. et al (2003)VE-cadherin-induced Cdc42 signaling regulates formation of membraneprotrusions in endothelial cells,” J. Biol. Chem. 278: 16230-36).Non-junctional VE-cadherin thus actively participates in inside-outsignaling at the plasma membrane, leading to the development ofendothelial membrane protrusions (Id).

During inflammation, inflammatory mediators increase vascularpermeability primarily by formation of intercellular gaps betweenendothelial cells of post-capillary venules. Spindler, V. et al (2010)“Role of GTPases in control of microvascular permeability,” Cardiovasc.Res. 87(2): 243-53). Adherens junctions of endothelial cells need to bedynamic when endothelial junctions transiently open to allow passage ofleukocytes from the blood into tissues. Rac1 and Cdc42 are the mainGTPases required for barrier maintenance and stabilization. RhoAnegatively regulates barrier properties (i.e., renders the barrier morepermeable) under both resting and inflammatory conditions (Id). RhoGTPases (RhoA, Rac1 and Cdc42) or Rap1 are known to regulate celladhesion in part by reorganization of the junction-associated corticalactin cytoskeleton (Id). Activated Cdc42 functions by counteracting thecanonical RhoA-mediated mechanism of endothelial hyperpermeability(Ramchandran, R. et al (2008) “Critical role of Cdc42 in mediatingendothelial barrier protection in vivo,” Am. J. Physiol. Lung Cell Mol.Physiol. 295: 363-69), while Rac1-mediated barrier destabilization inmicrovascular endothelium appears to be largely restricted to conditionsof enhanced endothelial cell migration and thus to be more closelyrelated to angiogenesis rather than to inflammation. (Spindler, V. et al(2010) “Role of GTPases in control of microvascular permeability,”Cardiovasc. Res. 87(2): 243-53)). Recent studies revealed that cAMPsignaling, which is well known to be barrier protective, enhancesbarrier functions in part via Rap1-mediated activation of Rac1 and Cdc42as well as by inhibition of RhoA. Moreover, barrier-stabilizingmediators directly activate Rac1 and Cdc42 or increase cAMP levels (Id).On the other hand, several barrier-disruptive components appear toincrease permeability by reduced formation of cAMP, leading to bothinactivation of Rac1 and activation of RhoA (Id).

The Cholesterol Biosynthesis Pathway

The mevalonate arm of the cholesterol biosynthesis pathway, whichincludes enzymatic activity in the mitochondria, peroxisome, cytoplasmand endoplasmic reticulum, starts with the consumption of acetyl-CoA,which occurs in parallel in 3 cell compartments (the mitochondria,cytoplasm, and peroxisome) and terminates with the production ofsqualene in the endoplasmic reticulum (Mazein, A. et al. (2013) “Acomprehensive machine-readable view of the mammalian cholesterolbiosynthesis pathway,” Biochemical Pharmacol. 86: 56-66). The followingare enzymes of the mevalonate arm:

Acetyl-CoA acetyltransferase (ACAT1; ACAT2; acetoacetyl-CoA thiolase; EC2.3.1.9) catalyzes the reversible condensation of two molecules ofacetylcoA and forms acetoacetyl-CoA (Id).

Hydroxymethylglutaryl-CoA synthase (HMGCS1 (cytoplasmic); HMGCS2(mitochondria and peroxisome); EC 2.3.3.10 catalyzes the formation of3-hydroxy-3-methylglutaryl CoA (3HMG-CoA) from acetyl CoA andacetoacetyl Co A (Id).

Hydroxymethylglutaryl-CoA lysase (mitochondrial, HMGCL; EC 4.1.3.4)transforms HMG-CoA into Acetyl-CoA and acetoacetate.

3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR; EC 1.1.34)catalyzes the conversion of 3HMG-CoA into mevalonic acid. This step isthe committed step in cholesterol formation. HMGCR is highly regulatedby signaling pathways, including the SREBP pathway (Id).

Mevalonate kinase (MVK; ATP: mevalonate 5-phosphotransferase; EC2.7.1.36) catalyzes conversion of mevalonate into phosphomevalonate(Id).

Phosphomevalonate kinase (PMVK; EC 2.7.4.2) catalyzes formation ofmevalonate 5-diphosphate from mevalonate 5-phosphate (Id).

Diphosphomevalonate decarboxylase (MVD; mevalonate (diphospho)decarboxylase; EC 4.1.1.33) decarboxylates mevalonate 5-diphosphate,forming isopentenyldiphosphate while hydrolyzing ATP (Id).

Isopentenyl-diphosphate delta-isomerases (ID11; ID12; EC 5.3.3.2)isomerize isopentenyl diphosphate into dimethylallyl diphosphate, thefundamental building blocks of isoprenoids (Id).

Farnesyl diphosphate synthase (FDPS; EC2.5.1.10; EC 2.5.1.1;dimethylallyltranstransferase) catalyzes two reactions that lead tofarnesyl diphosphate formation. In the first (EC 2.5.1.1 activity),isopentyl diphosphate and dimethylallyl diphosphate are condensed toform geranyl disphosphate. Next, geranyl diphosphate and isopentenyldiphosphate are condensed to form farnesyl diphosphate (EC 2.5.1.10activity) (Id).

Geranylgeranyl pyrophosphate synthase (GGPS1; EC 1.5.1.29; EC 2.5.1.10;farnesyl diphosphate synthase; EC 2.5.1.1;dimethylallyltranstransferase) catalyzes the two reactions of farnesyldiphosphate formation and the addition of three molecules of isopentenyldiphosphate to dimethylallyl diphosphate to form geranylgeranyldiphosphate (Id).

Farnesyl-diphosphate farnesyltransferase 1 (FDFT1; EC 2.5.1.21; squalenesynthase) catalyzes a two-step reductive dimerization of two farnesyldiphosphate molecules (C15) to form squalene (C30). The FDFT1 expressionlevel is regulated by cholesterol status; the human FDFT1 gene has acomplex promoter with multiple binding sites for SREBP-1a and SREBP-2(Id).

The sterol arms of the pathway start with Squalene and terminate withcholesterol production on the Bloch and Kandutsch-Russell pathways andwith 24 (S),25-epoxycholesterol on the shunt pathway (Id). The followingare enzymes of the sterol arms:

Squalene epoxidase (SQLE; EC 1.14.13.132, squalene monooxygenase)catalyzes the conversion of squalene into squalene-2,3-epoxide and theconversion of squalene-2,3-epoxide (2,3-oxidosqualene) into2,3:22,23-diepoxysqualene (2,3:22,23-dioxidosqualene). The firstreaction is the first oxygenation step in the cholesterol biosynthesispathway. The second is the first step in 24(S),25-epoxycholesterolformation from squalene 2,3-epoxide (Id).

Lanosterol synthase (LSS; OLC; OSC; 2,3-oxidosqualene:lanosterolcyclase; EC 5.4.99.7) catalyzes cyclization of squalene-2,3-epoxide tolanosterol and 2,3:22,23-depoxysqualene to 24(S),25-epoxylanosterol(Id).

Δ(24)-sterol reductase (DHCR24; 24-dehydrocholesterol reductase; EC1.3.1.72) catalyzes the reduction of the Δ-24 double bond ofintermediate metabolites. In particular it converts lanosterol into 24,25-dihydrolanosterol, the initial metabolite of the Kandutsch-Russelpathway and also provides the last step of the Bloch pathway convertingdesmosterol into cholesterol. Intermediates of the Bloch pathway areconverted by DHCR24 into intermediates of the Kandutsch-Russell pathway(Id).

Lanosterol 14-α demethylase (CYP51A1; cytochrome P450, family 51,subfamily A, polypeptide 1; EC 1.14.13.70) converts lanosterol into4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol and 24,25-dihydrolanosterolinto 4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol in three steps (Id).

Delta (14)-sterol reductase (TM7F2; transmembrane 7 superfamily member2, EC 1.3.1.70) catalyzes reactions on the three branches of thecholesterol and 24(S),25-epoxycholesterol pathways (Id).

Methylsterol monooxygenase 1 (MSM01; SC4MOL; C-4 methylsterol oxidase;EC 1.14.13.72) catalyzes demethylation of C4 methylsterols (Id).

Sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating (NSDHL;NAD(P) dependent steroid dehydrogenase-like; EC 1.1.1.170) participatesin several steps of post-squalene cholesterol and24(S),25-epoxycholeseterol synthesis (Id).

3-keto-steroid reductase (HSD17B7; 17-beta-hydroxysteroid dehydrogenase7; EC 1.1.1.270) converts zymosterone into zymosterol in the Blochpathway (Id).

3-β-hydroxysteroid-Δ(8), Δ(7)-isomerase (EBP; emopamil-binding protein;EC5.3.3.5) catalyzes the conversion of Δ(8)-sterols into Δ(7)-sterols(Id).

Lathosterol oxidase (SC5DL; sterol-C5-desaturase (ERG3 Δ-5-desaturasehomolog, S. cerevisiae-like; EC 1.14.21.6) catalyzes the production of7-dehydrocholesterol, 7-dehydrodesmosterol and24(S),25-epoxy-7-dehydrocholesterol (Id).

7-dehydrocholesterol reductase (DHCR7; EC 1.3.1.21) catalyzes reductionof the C7-C8 double bond of 7-dehydrocholesterol and formation ofcholesterol, and produces desmosterol from 7-dehydrodesmosterol and24(S),25-epoxycholesterol from 24(S),25-epoxy-7-dehydrocholesterol (Id).

Cytochrome P450, family 3, subfamily A, polypeptide 4 (CYP3A4;1,8-cineole 2-exo-monooxygenase; taurochenodeoxycholate 6α-hydroxylase;EC 1.14.13.97)) catalyzes the hydroxylation of cholesterol leading to25-hydroxycholesterol and 4β-hydroxycholesterol (Id).

Cholesterol 25-hydroxylase (CH25H; cholesterol 25-monooxygenase; EC1.14.99.38) uses di-iron cofactors to catalyze the hydroxylation ofcholesterol to produce 25-hydroxycholesterol, and has the capacity tocatalyze the transition of 24-hydroxycholesterol to 24,25-dihydroxycholesterol (Id).

Cytochrome P450, family 7, subfamily A, polypeptide 1 (CYP7A1;cholesterol 7-alpha-hydroxylase; EC 1.14.13.17) is responsible forintroducing a hydrophilic moiety at position 7 of cholesterol to form7α-hydroxycholesterol (Id).

Cytochrome P450, family 27, subfamily A, polypeptide 1 (CYP27A1; Sterol27-hydroxylase; EC 1.14.13.15) catalyzes the transition of mitochondrialcholesterol to 27-hydroxycholesterol and 25-hydroxycholesterol (Id).

Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase, EC1.14.13.98) catalyzes transformation of cholesterol into24(S)-hydroxycholesterol (Id).

Statins

The term “statin” as used herein refers to a competitive inhibitor ofHMG-CoA reductase in the mevalonate arm of the cholesterol biosynthesispathway. Exemplary statins include, without limitation, mevastatin,lovastatin, simvastatin, and pravastatin, which are fungal metabolites,and fluvastatin, atorvastatin, and verivastatin, which are syntheticcompounds. Statins exert their major effect—reduction of low densitylipoprotein cholesterol levels—through a mevalonic acid-like moiety thatcompetitively inhibits HMGCR by product inhibition. Higher doses of themore potent statins (e.g., atorvastatin and simvastatin) also can reducetriglyceride levels caused by elevated very low density lipoproteinlevels (Goodman & Gilman's The Pharmacological Basis of Therapeutics,Ed. Joel G. Hardman, Lee E. Limbird, Eds., 10th Ed., McGraw Hill, NewYork (2001), p. 984).

HMG-CoA reductase inhibition by the statins cerivastatin andatorvastatin has been shown to have a biphasic dose-dependent effect onangiogenesis that is lipid independent and associated with alterationsin endothelial apoptosis and VEGF signaling. (Weis, M. et al (2002)“Statins have biphasic effects on angiogenesis,” Cir. Res. 105: 739-45).Endothelial cell proliferation, migration, and differentiation of animmortalized human dermal microvascular endothelial cell line (HMEC-1)in vitro were enhanced at low statin concentrations (0.005 to 0.01μmol/L) but significantly inhibited at high statin concentrations (0.05to 1 μmol/L). Antiangiogenic effects at high concentrations wereassociated with decreased endothelial release of VEGF and increasedendothelial apoptosis and were reversed by geranylgeranyl pyrophosphate(GGP). GGP is required for the membrane localization of Rho familymembers. Other antiangiogenic effects of statins may include inhibitionof the expression or activity of monocyte chemoattractant protein-1,metalloproteinase and angiotensin-2, preproendothelin gene, and actinfilament and focal adhesion formation. In a zebrafish anti-angiogenicdrug screen, a number of statins (simvastatin, mevastatin, lovastatin,and rosuvastatin) were identified to inhibit angiogenesis in developingzebrafish embryos. The anti-angiogenic effect of rosuvastatin wasconfirmed in a mouse xenograft prostate cancer model. (Wang, C. et al,(2010) “Rossuvastatin, identified from a zebrafish chemical geneticscreen for anti-angiogenic compounds, suppresses the growth of prostatecancer,” Eur. Urol. 58: 418-26). In other murine models,inflammation-induced angiogenesis was enhanced with low-dose statintherapy (0.5 mg/kg/d) but significantly inhibited with highconcentrations of cerivastatin or atorvastatin (2.5 mg/kg/d). Despitethe fact that high-dose statin treatment was effective at reducing lipidlevels in hyperlipidemic apolipoprotein E-deficient mice, it impaired,rather than enhanced angiogenesis.

Prenylation

Prenylation is a class of lipid modification involving covalent additionof either farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoidsto conserved cysteine residues at or near the C-terminus of proteins(Zhang, F. L. and Casey, P J (1996) “Protein Prenylation: MolecularMechanisms and Functional Consequences,” Ann. Rev. Biochem. 65: 241-69).Prenylation promotes membrane interactions of prenylated proteins, andplays a major role in several protein-protein interactions involvingthem.

Both the 15-carbon isoprenoid FPP and the 20-carbon isoprenoid GGP areproducts of the MVA metabolic pathway; it follows that regulation ofHMGCR, FTase and GGTase-I, the key enzymes of the mevalonate pathway,can significantly affect the protein prenylation process. Zhang, F. L.and Casey, P J (1996) “Protein Prenylation: Molecular Mechanisms andFunctional Consequences,” Ann. Rev. Biochem. 65: 241-69).

Prenylated proteins can be grouped into two major classes: thosecontaining the CAAX motif and the so-called CC- or CxC-containingproteins. CAAX proteins are defined as a group of proteins with aspecific amino acid sequence at C-terminal that directs their posttranslational modification. Gao, J. et al (2009) “CAAX-box protein,prenylation process and carcinogenesis,” Am. J. Trans. Res. 1(3):312-25). C is cysteine residue, AA are two aliphatic residues, and Xrepresents any C-terminal amino acid depending on different substratespecificity. The CAAX proteins encompass a wide variety of moleculesthat include nuclear lamins (intermediate filaments), Ras and amultitude of GTP-binding proteins (G proteins), and several proteinkinases and phosphatases. Most CAAX proteins are found primarily at thecytoplasmic surface of cell membranes and are involved in a tremendousnumber of cellular signaling processes and regulatory events that playvarious roles in cell biological functions. These activities includecell proliferation, differentiation, nuclear stability, embryogenesis,spermatogenesis, metabolism, and apoptosis. The proteins that have aCAAX box at the end of the C-terminal always need a prenylation processbefore the proteins can be sent to plasma membrane or nuclear membraneand thereby exert their different functions.

Prenylation of CAAX proteins includes 3 steps: polyisoprenylation,proteolysis, and carboxyl methylation. Zhang, F. L. and Casey, P J(1996) “Protein Prenylation: Molecular Mechanisms and FunctionalConsequences,” Ann. Rev. Biochem. 65: 241-69). First, an isoprenoidlipid is attached to the CAAX box by a prenyltransferase, for example,FTase or GGTase-I. When the C terminal amino acid “X” is serine,methionine or glutamine, proteins are recognized by FTase, whereas aleucine at this position results in modification by GGTase I. FTase andGGTase-I recognize the CAAX box in the protein, and then add the15-carbon isoprenoid farnesyl pyrophosphate by FTase or the 20-carbonisoprenoid by GGTase-I to the cysteine residue of the CAAX box. Second,following prenylation, the aaX residues are cleaved by an endoprotease.Third, the carboxyl group of the modified cysteine is methylated by aspecific methyl transferase.

GGTase II transfers geranylgeranyl groups from GGPP to both cysteineresidues of CC- or CxC-containing proteins in a process mechanisticallydistinct from that of CAAX proteins. Additionally, proteins containingthe CxC motif are methylated at the C-terminal prenylcysteine, whereasCC-containing proteins are not.

HMGCR mediated GGPP biosynthesis regulates Cdc42 prenylation.(Eisa-Beygi S, Hatch G, Noble S, Ekker M, Moon T W (2013) “The3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway regulatesdevelopmental cerebral-vascular stability via prenylation-dependentsignaling pathway,” Dev Biol 373:258-266). Cdc42 regulates adherensjunction stability and endothelial barrier function.

Intracerebral Hemorrhage (ICH)

Spontaneous intracerebral hemorrhage (ICH) is a severe and debilitatingform of stroke that is most commonly due to hypertension, amyloidangiopathy, brain vascular malformations or secondary to medicationsincluding antiplatelet and anticoagulant drugs. Spontaneous ICHcomprises 10% of strokes and is associated with death or disability inmore than 50% of the approximately 90,000 patients affected each year inNorth America. (Roger V L, Go A S, Lloyd-Jones D M, Adams R J, Berry JD, Brown T M, et al (2011) “Heart disease and stroke statistics-2011update: a report from the American Heart Association,” Circulation123:e18-e209). Clinical studies also have disclosed a link betweencholesterol-lowering HMGCR inhibitors (statins) and increased risk ofICH. (Collins R, Armitage J, Parish S, Sleight P, Peto R: (2004)“Effects of cholesterol-lowering with simvastatin on stroke and othermajor vascular events in 20536 people with cerebrovascular disease orother high-risk conditions,” Lancet 363:757-767); Flaster M,Morales-Vidal S, Schneck M J, Biller J (2011) “Statins in hemorrhagicstroke,” Expert Rev Neurother 11:1141-1149; Goldstein L B, Amarenco P,Szarek M, Callahan A, III, Hennerici M, Sillesen H, et al (2008)“Hemorrhagic stroke in the Stroke Prevention by Aggressive Reduction inCholesterol Levels study,” Neurology 70:2364-2370). Another type ofbrain hemorrhage is brain microhemorrhages (BMH), which are small,usually multiple, ICHs. A systematic review found that 5% of healthyadults, 34% of patients with ischemic stroke and 60% of patients withnontraumatic ICH had BMH. (Cordonnier C, Klijn C J, van B J, Al-Shahi SR (2010) “Radiological investigation of spontaneous intracerebralhemorrhage: systematic review and trinational survey,” Stroke41:685-690). They are more common in patients with hypertension anddiabetes mellitus. Other than treatment of hypertension, there is noprophylactic treatment to prevent ICH or BMH.

Intracerebral Hemorrhage, Brain Microhemorrhages and Other Causes ofIntracerebral Hemorrhage

Spontaneous ICH accounts for 10% of strokes. There are about 90,000 peryear in the U.S. and Canada. Mortality is 30-50%. The most common causeis hypertension, and ICH due to hypertension can be partly reduced bytreating hypertension. However, other factors contribute to ICH fromhypertension, such as low serum cholesterol (Sutherland G R, Auer R N(2006) “Primary intracerebral hemorrhage,” J Clin Neurosci 13:511-517).The second main cause of ICH is amyloid angiopathy, for which there isno specific treatment. The pathophysiology of ICH from amyloid bloodvessels is unknown, although it is highly associated with amyloiddeposition in brain arteries and arterioles.

Brain microhemorrhages (BMH) are another form of ICH (Fisher M J (2013)“Brain regulation of thrombosis and hemostasis: from theory topractice,” Stroke 44:3275-3285). They are associated with increasingage, amyloid angiopathy, hypertension, ischemic/hemorrhagic stroke(mixed cerebrovascular disease) and Alzheimer disease. They are usuallyattributed to localized bleeding from tears in small arterioles butFisher proposed that they may be age-dependent, inflammation-mediatedleakage from small brain blood vessels (Id). This hypothesis issupported by BMH induced by lipopolysaccharide (LPS) in zebrafish andmice (FIGS. 5, 6 and 9) (Liu S, Vasilevko V, Cribbs D H, Fisher M (2013)“A mouse model of cerebral microhemorrhages,” Stroke 44:AWP297(Abstract)). Furthermore, patients with BMH are at increased risk of ICHand that risk is increased further if they take antiplatelet oranticoagulant drugs (Cordonnier C, Klijn C J, van B J, Al-Shahi S R(2010) “Radiological investigation of spontaneous intracerebralhemorrhage: systematic review and trinational survey,” Stroke41:685-690; Greenberg S M, Eng J A, Ning M, Smith E E, Rosand J (2004)“Hemorrhage burden predicts recurrent intracerebral hemorrhage afterlobar hemorrhage,” Stroke 35:1415-1420; Imaizumi T, Horita Y, HashimotoY, Niwa J (2004) “Dotlike hemosiderin spots on T2*-weighted magneticresonance imaging as a predictor of stroke recurrence: a prospectivestudy,” J Neurosurg 101:915-920). BMHs also are associated withcognitive impairment (Yakushiji Y, Noguchi T, Hara M, Nishihara M,Eriguchi M, Nanri Y, et al (2012) “Distributional impact of brainmicrohemorrhages on global cognitive function in adults withoutneurological disorder,” Stroke 43:1800-1805).

While statins reduce the long-term risk of myocardial infarction andischemic stroke, they increase the risk of ICH (Collins R, Armitage J,Parish S, Sleight P, Peto R (2004) “Effects of cholesterol-lowering withsimvastatin on stroke and other major vascular events in 20536 peoplewith cerebrovascular disease or other high-risk conditions,” Lancet363:757-767; Flaster M, Morales-Vidal S, Schneck M J, Biller J (2011)“Statins in hemorrhagic stroke,” Expert Rev Neurother 11:1141-1149;Goldstein L B, Amarenco P, Szarek M, Callahan A, III, Hennerici M,Sillesen H, et al (2008) “Hemorrhagic stroke in the Stroke Prevention byAggressive Reduction in Cholesterol Levels study,” Neurology70:2364-2370, Haussen D C, Henninger N, Kumar S, Selim M (2012) “Statinuse and microhemorrhages in patients with spontaneous intracerebralhemorrhage,” Stroke 43:2677-2681); Eisa-Beygi S, Wen X Y, Macdonald R L.(2014) “A Call for Rigorous Study of Statins in Resolution of CerebralCavernous Malformation Pathology.” Stroke 45(6):1859-61. According tothe American Heart Association guidelines, statins may not be indicatedin these patients. (Morgenstem L B, Hemphill J C, III, Anderson C,Becker K, Broderick J P, Connolly E S, Jr., et al (2010) “Guidelines forthe management of spontaneous intracerebral hemorrhage: a guideline forhealthcare professionals from the American Heart Association/AmericanStroke Association,” Stroke 41:2108-2129). Statins inhibit cholesterolsynthesis, and low serum cholesterol also is an independent risk factorfor ICH. (Sutherland G R, Auer R N (2006) “Primary intracerebralhemorrhage,” J Clin Neurosci 13:511-517).

Cerebral cavernous malformations (CCM) are the most common brainvascular malformation. They are found in 0.5% of the population and area cause of spontaneous ICH (Richardson B T, Dibble C F, Borikova A L,Johnson G L (2013) “Cerebral cavernous malformation is a vasculardisease associated with activated RhoA signaling,” Biol Chem 394:35-42).The hemorrhages tend to cluster in time so a drug that reduced this riskduring times of increased hemorrhage risk is actively being sought andis greatly needed (Barker F G, Amin-Hanjani S, Butler W E, Lyons S,Ojemann R G, Chapman P H, et al (2001) “Temporal clustering ofhemorrhages from untreated cavernous malformations of the centralnervous system,” Neurosurgery 49:15-24, Li Q, Mattingly R R (2008)“Restoration of E-cadherin cell-cell junctions requires both expressionof E-cadherin and suppression of ERK MAP kinase activation inRas-transformed breast epithelial cells,” Neoplasia 10:1444-1458). CCMmay be sporadic or inherited in association with loss-of-functionmutations in genes encoding 3 structurally distinct proteins, CCM1(KRIT1), CCM2 (Osmosensing scaffold for MEKK3 or OSM, MALCAVERIN, orMGC4607), and CCM3 (programmed cell death 10 (PDCD10)(Li D Y, WhiteheadK J (2010) “Evaluating strategies for the treatment of cerebralcavernous malformations,” Stroke 41:S92-S94). All 3 CCM proteins areinvolved in cytoskeleton and AJ and the mutations have to be inendothelial cells in order for CCMs to form. Mutations in CCM1 and CCM2lead to increased RhoA activity, which led to the hypothesis thatincreased RhoA activity affects the cell cytoskeleton and causesvascular instability, CCMs and possibly ICH/BMH in humans. Drugs thatinhibit RhoA activity, such as statins and fasudil, are theorized toreduce the risk of ICH based on data from mouse models of CCMs. (Li D Y,Whitehead K J (2010) “Evaluating strategies for the treatment ofcerebral cavernous malformations,” Stroke 41:S92-S94; Richardson B T,Dibble C F, Borikova A L, Johnson G L (2013) “Cerebral cavernousmalformation is a vascular disease associated with activated RhoAsignaling,” Biol Chem 394:35-42). This hypothesis is in contrast tostudies in zebrafish showing that statins impair vascular stability andgive rise to ICH/BMH (Eisa-Beygi S, Hatch G, Noble S, Ekker M, Moon T W(2013) “The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathwayregulates developmental cerebral-vascular stability viaprenylation-dependent signaling pathway,” Dev Biol 373:258-266). Theseeffects were shown to be due to defective prenylation of Rho GTPases,particularly Cdc42, a Rho GTPase involved in the regulation of vascularstability, leading to the question as to the cause of the discrepancybetween zebrafish and mouse models. In fact, statin treatment ofzebrafish induces cerebrovascular defects typified by leaky, dilatedcranial vessels with sluggish blood flow, which are analogous to CCMs.Without being limited by theory, it is hypothesized herein that thedifference is due to relative degrees of inhibition of RhoA and Cdc42,since the balance of vascular destabilizing RhoA to vascular stabilizingCdc42 may differ depending on dose and species.

Models of ICH and BMH

Zebrafish are emerging as useful model organism for large-scale,phenotype-based chemical and genetic screening. Zebrafish aregenetically very similar to humans, easy and fast to breed forhigh-throughput screening, transparent early on for easy imaging andrelatively easy to modify genetically. Some compounds discovered inzebrafish are effective in mammals and already in human studies(Peterson R T, Fishman M C (2011) “Designing zebrafish chemicalscreens,” Methods Cell Biol 105:525-541). Since the screening isperformed in vivo, general drug toxicity can be evaluated at the sametime as drug efficacy, allowing for a higher success rate as compared toan in vitro drug screens on cultured cells (Miscevic F, Rotstein O, WenX Y (2012) “Advances in zebrafish high content and high throughputtechnologies,” Comb Chem High Throughput Screen 15:515-521, 2012).Furthermore, an in vivo screen system such as zebrafish can measure theefficacy of the drug as well as its metabolites. The ability to performhigh throughput screening of compound libraries in zebrafish is anadvantage over testing compounds in rodents where high throughputscreening is not possible.

Several models of ICH/BMH have been identified in zebrafish. First,statins cause ICH/BMH in zebrafish (FIG. 7, 9) (Eisa-Beygi S, Hatch G,Noble S, Ekker M, Moon T W (2013) “The 3-hydroxy-3-methylglutaryl-CoAreductase (HMGCR) pathway regulates developmental cerebral-vascularstability via prenylation-dependent signaling pathway,” Dev Biol373:258-266). The mechanism is due to inhibition of protein prenylationsince ICH/BMH can also be induced by MO-induced depletion of the βsubunit of geranylgeranyltransferase 1 (GGTase 1, pggtlβ) and preventedby downstream metabolic rescue with the product of HMGCR, geranylgeranylpyrophosphate (GGPP, FIGS. 3, 4 and 8). GGPP is a 20 carbon lipidmolecule required for post-translational prenylation of Rho GTPaseproteins. The BMH temporal and spatial distribution is similar toICH/BMH seen in zebrafish bubblehead (bbhm292) and redhead (rhdmi149)mutants. (Buchner D A, Su F, Yamaoka J S, Kamei M, Shavit J A, Barthel LK, et al (2007) “pak2a mutations cause cerebral hemorrhage in redheadzebrafish,” Proc Natl Acad Sci USA 104:13996-14001; Butler M G, Gore AV, Weinstein B M (2011) “Zebrafish as a model for hemorrhagic stroke,”Methods Cell Biol 105:137-161; Liu J, Fraser S D, Faloon P W, Rollins EL, Vom B J, Starovic-Subota O, et al (2007) “A betaPix Pak2a signalingpathway regulates cerebral vascular stability in zebrafish,” Proc NatlAcad Sci USA 104:13990-13995).

Bubblehead (bbh^(m292)) develops ICH and brain edema 36 to 52 hourspostfertilization (hpf) whereas redhead (rhd^(mi149)) develops ICH 2 to3 days postfertilization. The bbh^(m292) mutation is in the βpix(pak-interacting exchange factor β) gene, whereas the rhd^(mi149)mutation is in pak2a (p21 protein [Cdc42/Rac]-activated kinase 2a) gene.These genes encode proteins that regulate activity of Rho GTPases, Racand Cdc42. That both of these changes are associated with ICH/BMH isconsistent with Rac and Cdc42 requiring GGTase 1-mediated prenylation.GGTase 1 post-translationally modifies Rac and Cdc42 by adding amevalonate-derived GGPP which is required to activate these GTPases.(Peterson Y K, Kelly P, Weinbaum C A, Casey P J (2006) “A novel proteingeranylgeranyltransferase-I inhibitor with high potency, selectivity,and cellular activity,” J Biol Chem 281:12445-12450) There are alsozebrafish mutants corresponding to the orthologous human CCM1, CCM2 andCCM3 genes (Butler M G, Gore A V, Weinstein B M (2011) “Zebrafish as amodel for hemorrhagic stroke,” Methods Cell Biol 105:137-161). Thesedevelop cardiac dilation and progressively enlarged, dilated bloodvessels and it has been suggested the genes have similar function inboth species. No ICH phenotype is described in these zebrafish mutantsbut combined MO-induced reduction in a Ras GTPase effector protein, raplb and zebrafish ccml did cause ICH (Gore A V, Lampugnani M G, Dye L,Dejana E, Weinstein B M (2008) “Combinatorial interaction between CCMpathway genes precipitates hemorrhagic stroke,” Dis Model Mech1:275-281). In mice, the gene defects for CCMs are suggested to berequired in endothelial cells in order for malformations to develop(Chan A C, Li D Y, Berg M J, Whitehead K J (2010) “Recent insights intocerebral cavernous malformations: animal models of CCM and the humanphenotype,” FEBS J 277:1076-1083).

Mouse models of ICH include direct injection of blood into the brain, orinjection of elastase, which degrades vascular collagen and causesbleeding. These models would not be useful for detecting therapeuticagents that stabilize the vasculature. There are two models ofspontaneous ICH in mice. One is a model of acute and chronichypertension induced by a combination of angiotensin 2 and NOSinhibition in mice (Wakisaka Y, Chu Y, Miller J D, Rosenberg G A,Heistad D D (2010) “Spontaneous intracerebral hemorrhage during acuteand chronic hypertension in mice,”. J Cereb Blood Flow Metab 30:56-69).The mechanism of ICH in hypertension, however, may differ from what weare investigating with statins and CCM genes.

A second model of BMH involves transgenic mice (e.g. Tg2576) thatspontaneously overexpress P-amyloid, mimicking cerebral amyloidangiopathy (Herzig M C, Winkler D T, Burgermeister P, Pfeifer M, KohlerE, Schmidt S D, et al. Abeta is targeted to the vasculature in a mousemodel of hereditary cerebral hemorrhage with amyloidosis. Nat Neurosci.2004; 7(9):954-60, Fisher M, Vasilevko V, Passos G F, Ventura C, QuiringD, Cribbs D H. Therapeutic modulation of cerebral microhemorrhage in amouse model of cerebral amyloid angiopathy. Stroke. 2011;42(11):3300-3). There are other similar models. (Alharbi B M, Tso M K,Macdonald R L. (2016) Animal models of spontaneous intracerebralhemorrhage. Neurol Res 38:448-455). The limitation is that it takes upto 2 years for animals to develop BMH.

LPS has been used to induce BMH in mice (Tang, A T, et al, “EndothelialTLR4 and the microbiome drive cerebral cavernous malformations,” Nature(2017) 545 (7654): 305-310. Doi: 10.1038/nature22075; Liu S, VasilevkoV, Cribbs D H, Fisher M (2013) “A mouse model of cerebral,” Stroke44:AWP297 [Abstract]); Liu S, Grigoryan M M, Vasilevko V, Sumbria R K,Paganini-Hill A, Cribbs D H, et al. (2014) “Comparative analysis of H &E and prussian blue staining in a mouse model of cerebral microbleeds.”J Histochem Cytochem. 62:767-773). LPS or vehicle (phosphate bufferedsaline [PBS]) was injected at baseline and again at 24 hours, and themice were sacrificed 2 days after the first injection (FIGS. 6 and 9).When the brains were examined, multiple small fresh hemorrhages werefound in mice treated with LPS, as was increased blood brain barrierpermeability. It has been suggested that this model might be useful tostudy mechanisms of and interventions for BMH.

Anti-β3 Integrin Mouse Model of Intracerebral Hemorrhage (ICH)

The integrin αIIbβ3 is the most abundant glycoprotein on platelets. Theβ3 subunit also is coexpressed with the αV subunit (i.e., αVβ3) onproliferating endothelial cells (ECs) during angiogenesis (Yougbare, I.et al., “Maternal anti-platelet β3 integrins impair angiogenesis andcause intracranial hemorrhage,” (2015) J. Clin. Invest. 125(4): 1545-56citing Brooks, P C et al, “Requirement of vascular integrin alpha v beta3 for angiogenesis,” Science (1994) 264 (5158): 569-71; Brooks, P C etal, “Integrin αvβ3 antagonists promote tumor regression by inducingapoptosis of angiogenic blood vessels,” (1994) Cell 79(7): 1157-64; DiQ, et al, “Impaired cross-activation of β3 integrin and VEGFR-2 onendothelial progenitor cells with aging decreases angiogenesis inresponse to hypoxia,” Intl J. Cardiol. (2013) 168(3): 2167-76; Stupack,D C, Cheresh, D A, “Integrins and angiogenesis,” Curr. Top. Dev. Bio.(2004) 64: 207-38. Several studies have demonstrated that β3 plays animportant role in angiogenesis. For example, it has been shown that αVb3was required for angiogenesis (Id. diting Brooks, P C et al,“Requirement of vascular integrin alpha v beta 3 for angiogenesis,”Science (1994) 264 (5158): 569-71), and that αVb3 antagonists promotedtumor regression by inducing apoptosis of angiogenic blood vessels (Id.citing Brooks, P C et al, “Integrin αvβ3 antagonists promote tumorregression by inducing apoptosis of angiogenic blood vessels,” (1994)Cell 79(7): 1157-64). Evidence has also shown that integrin αVβ3cooperated with VEGFR2 in pro-angiogenic signaling (Id., citingRobinson, S D, et al, “αvβ3 integrin limits the contribution ofneuropilin-1 to vascular endothelial growth factor-inducedangiogenesis,” J. Biol. Chem. (2009) 284(49): 33966-81; Soldi, R. etall, “Role of αvβ3 integrin in the activation of vascular endothelialgrowth factor receptor-2,” EMBO J. (1999) 18(4): 882-92) and that AKTphosphorylation was essential in VEGF-mediated post-natal angiogenesis(Id. citing Kitamura, T et al, “Regulation of VEGF-mediated angiogenesisby the Akt/PKB substrate Girdin,” Nat. Cell Biol. (2008): 10(3):329-337).

An established murine model of fetal and neonatal autoimmunethrombocytopenia (FNAIT) has been used to investigate the mechanism ofICH in affected fetuses and neonates (Id., citing Chen, P. et al.,“Animal model of fetal and neonatal immune thrombocytopenia: role ofneonatal Fc receptor in the pathogenesis and therapy,” Blood (2010) 116(18): 3660-68; Li, C. et al, “The maternal immune response to fetalplatelet GpIbα causes frequent miscarriage in mice that can be preventedby intravenous IgG and anti-FcRn therapies,” J. Clin. Invest. (2011)121(11): 4537-47; Ni H, et al, “A novel murine model of fetal andneonatal alloimmune thrombocytopenia: response to intravenous IgGtherapy,” Blood (2006) 107(7): 2976-83). Itgb3^(−/−) and Gp1ba^(−/−)mice (referred to hereinafter as β3^(−/−) and GPIbα^(−/−)) weretransfused with WT platelets to mimic exposure to β3 or to GPIbα duringconception. Id. Anti-β3 or anti-GPIbα antibodies were detected; theseimmunized mice were subsequently bred with WT males. Id. Similarseverity of thrombocytopenia in the heterozygote (−/+) neonatesdelivered from immunized β3^(−/−) and GPIBα^(−/−) mice was found. Id.ICH was found in the β3^(−/−) fetuses starting around EC15.5 as well asin neonates using a high-frequency ultrasound imaging system to detectin utero ICH in pregnant mice, and performing H & E staining of brainsections. Id. Hemorrhage was observed in different areas of the brain,and the frequency of ICH increased in fetuses in accordance with thenumber of material immunizations. Id. ICH was never found inanti-GPIbα-mediated FNAIT fetuses or neonates. Id.

The following experiments showed that anti-β3 antibodies, but notanti-GPIbα antibodies or thrombocytopenia alone, were the cause of ICH.To confirm that ICH was indeed antibody mediated, β3^(−/−) andGPIBα^(−/−) neonates delivered from naïve mice were passively injectedwith antisera at P2. Postnatal injection of anti-β3 sera into β3^(−/−)neonates induced ICH, but anti-GPIbα sera did not induce any ICH inGPIBα^(−/−) neonates (P<0.01). Id. To further determine whetherplatelet-mediate cytotoxicity (Id. Citing Nieswandt, B. et al.,“Identification of critical antigen-specific mechanisms in thedevelopment of immune thrombocytopenic purpura in mice,” Blood (2000)96(7): 2520-27; Nieswandt, B. et al, “Targeting of platelet integrin011433 determines systemic reaction and bleeding in murinethrombocytopenia regulated by activating and inhibitory FcγR,” IntlImmunol. (2003) 15(3): 341-49) might be involved in the mechanism ofICH, anti-β3 sera were injected into αIIb integrin-deficient pups thatdid not express αIIbβ3 integrin on their platelets. Id. ICH was observedin Itga2b^(−/−) pups with normal platelet counts, and postnatalinjection of anti-β3 sera into β3^(−/−) neonates failed to induce ICHand impair retinal vascular development in these antigen-negative pups.Id.

Mouse models that combine Ccm heterozygotes on a background ofhomozygous deletion of the mismatch repair complex protein Msh2(<Ccm1^(+/−)Msh^(−/−) and Ccm2+/‘Msh^(−/−)) develop CCMs. (McDonald D A,Shi C, Shenkar R, Stockton R A, Liu F, Ginsberg M H, et al, (2012)“Fasudil decreases lesion burden in a murine model of cerebral cavernousmalformation disease,” Stroke 43:571-574. Another important gene isRap1b, mouse mutants of which develop normally until embryonic day 12.5,at which point 50% die due to hemorrhage (Id); Chrzanowska-Wodnicka M(2013) “Distinct functions for Rap1 signaling in vascular morphogenesisand dysfunction,” Exp Cell Res 319:2350-2359). Subphenotypic levels ofreduction of ccml and rap lb in zebrafish cause brain hemorrhage.

3. Mechanisms of ICH and BMH

In patients with hypertension, the cause of ICH is arteriolosclerosis ofthe small penetrating arteries that tend to arise from large conductingcerebral arteries. (Auer, R N, Sutherland, G R (2005) “Primaryintracerebral hemorrhage: pathophysiology,” Can. J. Neurol. Sci. 32Suppl. 2: 3-12). The only currently available treatment is prophylactictreatment of hypertension. Guidelines for management of patients oncethey have a hypertensive ICH are published, and recommend surgicalevacuation of space-occupying cerebellar ICH and general medicalsupportive care. (Hemphill J C, 3rd, Greenberg S M, Anderson C S, BeckerK, Bendok B R, Cushman M, et al. Guidelines for the management ofspontaneous intracerebral hemorrhage: A guideline for healthcareprofessionals from the American Heart Association/American StrokeAssociation. Stroke. 2015; 46:2032-2060).

There are different theories as to why statins increase the risk of ICH.For example, statin-associated ICH and other types of ICH/BMH may be dueto defects in the HMGCR pathway (Eisa-Beygi S, Hatch G, Noble S, EkkerM, Moon T W (2013) “The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)pathway regulates developmental cerebral-vascular stability viaprenylation-dependent signaling pathway,” Dev Biol 373:258-266).Inhibition of HMGCR or of other downstream molecules such asgeranylgeranyltransferase 1 (GGTase 1) causes ICH/BMH in zebrafishembryos (Id). Flaster and colleagues suggested that statins could causechanges in platelets or in the interactions between clotting andfibrinolytic cascades that could promote ICH, although there is noevidence for this thus far (Flaster M, Morales-Vidal S, Schneck M J,Biller J (2011) “Statins in hemorrhagic stroke,” Expert Rev Neurother11:1141-1149).

There are at least 2 mutations that cause ICH in zebrafish. Bubbleheadis a loss of function mutation of βpix (p isoform of the p21-activatingkinase (Pak)-interacting exchange factor) (Liu J, Zeng L, Kennedy R M,Gruenig N M, Childs S J (2012) “betaPix plays a dual role in cerebralvascular stability and angiogenesis, and interacts with integrinalphavbeta8,” Dev Biol 363:95-105). βpix regulates vascular stabilityand βpix mutation is the cause of vascular fragility and ICH in thebbh^(m292) mutant. (Liu J, Fraser S D, Faloon P W, Rollins E L, Vom B J,Starovic-Subota O, et al (2007) “A betaPix Pak2a signaling pathwayregulates cerebral vascular stability in zebrafish,” Proc Natl Acad SciUSA 104:13990-13995). βpix also is involved in focal adhesion complexes,which contain integrins and cadherins (Frank S R, Hansen S H (2008) “ThePIX-GIT complex: a G protein signaling cassette in control of cellshape,” Semin Cell Dev Biol 19:234-244). Another zebrafish mutant,redhead, is rhd^(mi149) that has a mutation pak2a (p21 protein[Cdc42/Rac]—activated kinase 2a) Liu J, Fraser S D, Faloon P W, RollinsE L, Vom B J, Starovic-Subota O, et al (2007) “A betaPix Pak2a signalingpathway regulates cerebral vascular stability in zebrafish,” Proc NatlAcad Sci USA 104:13990-13995). pak2a is a kinase acting downstream ofCdc42 and Rac, and may be involved in a complex with βPix, paxillin andGIT1. Since Cdc42 also is required for interaction of VE-cadherin andthe actin cytoskeleton, the defect in the bbh^(m292) mutant could alsobe due to defects in this latter interaction. In mice, germ-line orendothelial cell specific Pak2 knockout is embryonic lethal likely dueto impaired blood vessel formation. (Radu M, Semenova G, Kosoff R,Chemoff J (2014) “PAK signalling during the development and progressionof cancer,” Nat Rev Cancer 14:13-25). Both are transmembrane proteinsthat connect the intracellular cytoskeleton to the extracellular matrix(Frank S R, Hansen S H (2008) “The PIX-GIT complex: a G proteinsignaling cassette in control of cell shape,” Semin Cell Dev Biol19:234-244; van der Flier A, Sonnenberg A (2001) “Function andinteractions of integrins,” Cell Tissue Res 305:285-298). They areimportant in angiogenesis and vascular stability. For example,homozygous integrin α_(v) or β₈ null mice die perinatally or before fromICH (Zhu J, Motejlek K, Wang D, Zang K, Schmidt A, Reichardt L F (2002)“β₈ integrins are required for vascular morphogenesis in mouse embryos,”Development 129:2891-2903). Targeted inactivation of VE-cadherin andtruncation of the β-catenin-binding cytosolic domain of VE-cadherin inmice induces endothelial cell-specific apoptosis, in addition todefective remodeling and maturation of the vasculature and earlylethality (Carmeliet P, Lampugnani M G, Moons L, Breviario F, CompemolleV, Bono F, et al (1999) “Targeted deficiency or cytosolic truncation ofthe VE-cadherin gene in mice impairs VEGF-mediated endothelial survivaland angiogenesis,” Cell 98:147-157). Most recently, we demonstrated thatby delivering anti-β₃ integrin antibody into pregnant mice could induceICH in mouse embryos and neonatal mice. (Yougbaré I, Lang S, Yang H,Chen P, Zhao X, Tai W S, Zdravic D, Vadasz B, Li C, Piran S, Marshall A,Zhu G, Tiller H, Killie M K, Boyd S, Leong-Poi H, Wen X Y, Skogen B,Adamson S L, Freedman J and Ni H (2015) “Maternal anti-platelet β3integrin antibodies impair angiogenesis and cause intracranialhemorrhage in fetal and neonatal alloimmune thrombocytopenia,” J.Clinical Investigation (JCI), 125:1545-56). Morpholino-induced reductionof cdh5, the zebrafish homologue of the VE-cadherin gene in humans,causes vascular instability, defective lumen formation and ICH by 48 hpf(Montero-Balaguer M, Swirsding K, Orsenigo F, Cotelli F, Mione M, DejanaE (2009) “Stable vascular connections and remodeling require fullexpression of VE-cadherin in zebrafish embryos,” PLoS ONE 4:e5772). Itis known that integrins are linked to βPix in focal adhesions byproteins including G protein-coupled receptor kinase interacting target(GIT1), which is an Arf GTPase activating protein (GAP) (Liu J, Zeng L,Kennedy R M, Gruenig N M, Childs S J (2012) “betaPix plays a dual rolein cerebral vascular stability and angiogenesis, and interacts withintegrin alphavbeta8,” Dev Biol 363:95-105). Mice without GIT1 haveincreased pulmonary vascular density and pulmonary hemorrhage. Inzebrafish GIT1 is initially ubiquitously expressed but it becomesrestricted to the head by 48 hpf. Knock down of GIT1 expression with MOscauses increases in ICH. Liu, et al., conducted a series of experimentsin zebrafish that suggest that βPix interacts with α_(v) and β₈integrins and GIT1 to stabilize the cerebral vasculature, and inhibitingexpression of any of the components causes ICH (Liu J, Zeng L, Kennedy RM, Gruenig N M, Childs S J (2012) “betaPix plays a dual role in cerebralvascular stability and angiogenesis, and interacts with integrinalphavbeta8,” Dev Biol 363:95-105).

CCM1, 2 and 3 may form a multiprotein complex that also includesintegrin β1-binding protein (ICAP-1), the GTPases Rac and Rap1 and MAPKkinase kinase MEKX3 Chrzanowska-Wodnicka M (2013) “Distinct functionsfor Rap1 signaling in vascular morphogenesis and dysfunction,” Exp CellRes 319:2350-2359; Liu J, Fraser S D, Faloon P W, Rollins E L, Vom B J,Starovic-Subota O, et al (2007) “A betaPix Pak2a signaling pathwayregulates cerebral vascular stability in zebrafish. Proc Natl Acad SciUSA 104:13990-13995). This complex modulates adherens junctions byinteracting with β catenin and VE-cadherin mainly in endothelial cells(but probably other perivascular cells) and achieving vascularstability. Effects of knockouts of the ccm genes in zebrafish and miceare not fully explored but show that embryonic germ-line knockouts tendnot to resemble the human phenotype, although in some cases,conditional, endothelial cell specific knockouts do. In zebrafish,knockdown of rap1b leads to ICH, but also sub-phenotype levels ofreduction in rap1b combined with ccm1 leads to ICH.

It is likely that the cellular localization and other factors influencethe effect of GTPase signaling on vascular stability and ICH/BMH becausein mice with mutations in germ-line or endothelial cell-specificmutations in CCM2, statin inhibition of HMGCR, which reduces Rho GTPaseprenylation, improves endothelial integrity and prevents the increasedvascular permeability. In zebrafish, however, inhibition of HMGCR withstatins or mutation and MO-induced loss of function of proteins thatactivate Rho GTPase signaling increase vascular permeability (Liu J,Fraser S D, Faloon P W, Rollins E L, Vom B J, Starovic-Subota O, et al(2007) “A betaPix Pak2a signaling pathway regulates cerebral vascularstability in zebrafish,” Proc Natl Acad Sci USA 104:13990-13995). Thediscrepancy is likely due to cell-specific effects, age of theorganisms, species differences, differential effects on RhoA and cdc42(that tend to have opposing effects, RhoA destabilizing cdc42stabilizing vasculature) or differences between wild-type and CCM2animals.

Statement of the Problem

Intracerebral hemorrhage, BMH and cavernous malformations share commonelements of vascular instability in blood vessels in the brain that leadto intracranial hemorrhage, to brain injury and to death and disability.Intracerebral hemorrhage is the most lethal and devastating type ofstroke. There is no pharmacologic treatment available to reduce this andthere is a high unmet medical need. Therefore, a need exists for apharmaceutical composition comprising a therapeutic amount of a vascularstabilizing agent, in some embodiments formulated as a sustained releasepreparation, that when administered, is effective to prevent or reducethe incidence ICH, BMH and ICH from various causes including braincavernous malformations.

SUMMARY OF THE INVENTION

According to one aspect, the described invention provides a method forreducing incidence of vascular leakage comprising administering apharmaceutical composition containing a small molecule therapeuticcompound, a therapeutic amount of which is effective to reduce incidenceof bleeding in the brain by at least 30% relative to a control.

According to one embodiment, the small molecule therapeutic compound isselected from the group consisting of artemether or a derivative ofartemether. According to another embodiment, the derivative ofartemisinin is dihydroartemisinin, artemisinin, or artesunate. Accordingto another embodiment, the small molecule therapeutic compound isselected from the group consisting of benidipine, lacidipine,ethynylestradiol or triptolide.

According to one embodiment, the vascular leakage is induced by astatin, by a lipopolysaccharide, or both. According to anotherembodiment, the statin is atorvastatin.

According to one embodiment, the vascular leakage is a spontaneousintracerebral hemorrhage. According to another embodiment, thespontaneous intracerebral hemorrhage occurs in association with amutation of one or more genes selected from beta-pix, Pak2a, cdh5, ccm1,ccm2, ccm3, Rap1b, Pggt1b, Hmgcrb, and beta3 integrin.

According to one embodiment, the vascular leakage includes a brainmicrohemorrhage. According to another embodiment, the brainmicrohemorrhage occurs in association with administration of a statin.

According to one embodiment, the vascular leakage comprises a brainvascular malformation. According to another embodiment, the brainvascular malformation is a cerebral cavernous malformation.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor.

Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIG. 1 shows the results of experiments using an atorvastatin-inducedintracerebral hemorrhage (ICH) model in zebrafish for chemicalscreening. Panel (A) is a schematic diagram showing the molecularpathway where statins act. Panels B-G: ICH was induced by application of1 μM atorvastatin at 2 hours post fertilization (hpf) of embryos fromadult wild type or Tg (flk-1:eGFP) and Tg (gata-1:DsRed) zebrafish, andarrayed into 96-well plates that contain the drug compounds. Panels B, Dand F (embryos treated with DMSO control); panels C, E and G, (embryostreated with atorvastatin). Panels B, D and F show no extravasations ofred blood cells in vehicle DMSO-treated control embryos. Atorvastatintreated embryos show hemorrhage in the brain (≈80% panels C and G), andincreased junction between endothelial cells (compare Panel E to panelD). Panel H is a schematic showing the scheme of the screening process.Panels I to L show EC50 experiments for four compounds from the ARTfamily, two of which were identified from the NCC library. Data isexpressed as mean±SEM from 3 to 4 experiments. ARM, artemether; DHA,dihydro-artemisinin; ARS, artemisinin; ART, artesunate.

FIG. 2 shows inhibition of brain hemorrhage induced by 1 μM atorvastatinin zebrafish for four active compounds identified from NCC libraries.Plots are % hemorrhage (y axis) vs. drug concentration (Ig nmol/L)(x-axis). EC50 is concentration of the drug at 50% of efficacy. Data isexpressed as mean±SEM from 3 to 4 experiments. B is benidipine; E isethynylestradiol; L is lacidipine, and T is triptolide.

FIG. 3 shows the relationship between the HMGCR-mediated metabolicpathway, the Rho GTPase (Cdc42)-cadherin signaling pathway, and cerebralcavernous malformation (Ccm) pathways in zebrafish. By inhibitingHMG-CoA, statin treatment may lead to vascular instability and brainhemorrhage in zebrafish through the CDC42-cadherin pathway. ART familycompounds are shown to be effective in rescuing brain hemorrhage causedby genetic knockdown of several key molecules of this pathway (e.g.,pak2, βpix). This suggests that ART compounds act on a downstream targetthat is vital for vascular stability in the brain.

FIG. 4 shows examples of results from drug efficacy assays in thezebrafish bbh model for two compounds, ART, artesunate; and ARM,artemether. Results are plotted as percent hemorrhage (y-axis) vs. log(drug nmol/L); n=30 larvae per condition.

FIG. 5 shows examples of results from drug efficacy assays in zebrafishhmgcrb morphants using artesunate (ART), and artemether (ARM). Resultsare plotted as percent hemorrhage (y-axis) vs. log (drug nmol/L);n=15-20 larvae per condition.

FIG. 6 shows mRNA changes upon treatment with atorvastatin (ATV) andwith atorvastatin plus artemether (ATV+ARM). qRT-PCR analysis was usedto evaluate the mRNA level of gene expression of VE-cadherin (panel A),β3-integrin (panel B), and CCM3 (Panel C), in zebrafish treated with 1μM atorvastatin (ATV), with ATV plus 500 nmol/L of artemether (ARM) asshown, n=3.

FIG. 7 shows the results of toxicity assays for artesunate (ART (GMP))on heart beat, blood flow and heart edema. Heart beat and blood flowwere ranked from 3 (normal heart beat or blood flow) to 0 (no heart beator blood flow). Cardiac edema was ranked from 0 (normal heart withoutedema) to −3 (severe cardiac edema). TC50 is the concentration of thedrug at 50% of maximum toxicity. Data is expressed as mean±SEM from 3experiments.

FIG. 8 shows that artemether (ARM) rescues LPS-induced brain microbleedsin mice. Panel A shows data from a stereomicroscope count of surfacemicrobleeds in brains from LPS treated mice (n=8) orLPS+artemether-treated mice (n=8). The left panel shows representativeimages from each of the two groups; arrows indicate microbleeds. Theright panel shows a statistical analysis (*P<0.05, two-tailed t-testwith Welch correction); data is expressed as mean±SD. As compared to LPStreated animals, brains from ARM treated mice showed a robust reductionin total surface microbleeds. Panel B shows data from quantification ofmicrobleeds on brain slices stained by hematoxylin and eosin. The leftpanel shows representative images of stained brain slices from each ofthe two groups; the arrows indicate microbleeds on the slices; the rightpanel chart shows a statistical analysis of microbleeds count (**P<0.01,unpaired two-tailed t-test with Welch's correction). Data is expressedas mean±SD, n=8 for both LPS treated and LPS+ARM treated groups. Similarto the surface microbleed counts, ARM treatment significantly reducedthe total number of microbleeds inside the mouse brains.

FIG. 9 shows that artemether (ARM) rescues microbleeding induced bylipopolysaccharide (LPS) in mice. Panel (A) shows representative 3-Dreconstructed images from T2*−Weighted Gradient Echo (GRE) MRI sequencewith high resolution detection, in mouse cerebral cortex two days afterLPS injection or in LPS+ARM treated brains. Arrows indicate microbleeds.(B) is a bar graph showing the number of microbleedings per brain in avehicle control group and a group treated with artemether (ARM).Quantification of total microbleed volume was calculated usingsemi-automated software (Display), normalized to total brain volume, andexpressed as total voxel in 10000 counts. Data is expressed as mean±SD(*P<0.05, two-tailed t-test with Welch correction); n=8 for both LPStreated and LPS+ARM treated groups, 2 for naïve controls.

FIG. 10 shows that artemether (ARM) reduces ICH in an anti-β3 integrinmouse model of intracerebral hemorrhage. Panel A shows representativeraw T2*-Weighted Gradient Echo (GRE) MRI images of brains of miceinjected with anti-β3 integrin serum at post-natal day 2 alone (left) ortreated with ARM (right). Panel B shows paraffin-embedded blocks ofcoronally-cut whole brains from anti-β3 serum injected mice without(left) or with (right) ARM treatment, respectively. Panel C showsquantification of frequency of intracerebral hemorrhage in mice injectedwith anti-β3 integrin serum alone or with ARM treatment. Data isexpressed as mean±SD (**P<0.01, two-tailed t-test with Welchcorrection); n=29 and 24 for anti-β3 integrin serum injected micewithout or with ARM treatment, respectively.

FIG. 11 shows a plot of blood hemoglobin (g/dl) (y-axis) for controls,and for mice treated with artemether (ARM) (Treatment Dose, and 4×Treatment Dose). ARM treatment for 3 days did not cause anemia in mice.Bloods were tested for hemoglobin concentration after ARM treatment.Blood hemoglobin concentration was assessed using Drabkins' method.Spectrophotometer data was compared to a standard curve from standardcyanmethemoglobin concentrations. The control group received no drug.The Treatment Dose group received 3 days injection of low dose ARM (25mg/kg); 4× Treatment Dose group received 3 days injection of high doseARM (100 mg/kg). Data is expressed as mean±SD (nsP>0.05, one-way ANOVA,n=4).

FIG. 12 shows the pharmacological and genetic induction of loss ofcerebrovascular stabilization in developing zebrafish. (A) Schematicrepresentation of genetic pathways involved in cerebrovascularstabilization. (B) Schematic representation of VE-cadherin-mediatedcell-cell adhesion regulated in part by Cdc42. (C) Schematicrepresentation illustrating that unprenylated Cdc42 remains inactive(GDP-bound) and associated with guanine nucleotide dissociationinhibitor (GDI). (D) Photograph depicting un-injected embryos. (E)Photograph depicting embryos injected with MOs targeting hmgcrb (E). (F)Photograph depicting embryos injected with MOs targeting pggt1b. (G)Photograph depicting embryos injected with MOs targeting βpix. (H)Photograph depicting embryos injected with MOs targeting pak2a. Arrowsdenote the sites of abnormal accumulation of blood. (I, K) arerepresentative photomicrographs of Tg(fli1:EGFP);(gata-1:DsRed) embryosincubated in DMSO. (J, L) are representative photomicrographs ofTg(fli1:EGFP);(gata-1:DsRed) embryos incubated in atorvastatin. Thearrows in (J) indicate areas where stagnant DsRed-positive erythrocyteaccumulation is observed. The arrows in (L) denote distended cerebralvessels in the same fish. (M, P) are photographs depicting hemorrhagesassociated with the fragmentation of the underlying vasculature. (N, O)depict representative bright-field photomicrographs of Tg (fli1:EGFP);(gata-1:DsRed) embryos incubated in DMSO. (Q, R) depict representativebright-field photomicrographs of Tg (fli1:EGFP);(gata-1:DsRed) embryosincubated in atorvastatin. The asterisk denotes the hemorrhage and theblack dotted area shows the field of interest. Z-stack projections ofthe black dotted area in the same Tg(fli1:EGFP);(gata-1:DsRed) embryos.The white asterisk denotes DsRed-positive erythrocytes and the whitearrows show regions where vascular disintegration is observed. Anterioris to the left as shown in (I-R).

FIG. 13 shows the HMGCR molecular pathway that leads to vascularstability in zebrafish. Panels A & B: Schematics illustrating stable ECjunctions are maintained by a Cdc42-dependent and VE-cadherin-mediatedcell-cell adhesion. Panel C shows that splice-inducing morpholinosdesigned against cdh5 induced intracerebral hemorrhage in zebrafish at36-48 hpf (lateral images are shown).

FIG. 14 Panels (A-B) shows that artesunate dose-dependently rescueshemorrhage phenotype induced by morpholinos targeting membrane stabilityof brain vessels in zebrafish. (A) Schematic diagram showing the targetsites of the three morpholinos studied. (B) Artesunate dose-dependentlyrescues all three morpholinos-induced brain hemorrhage in zebrafish.Panels (C-D) Artesunate rescues the ICH phenotype underlying thebbh^(m292) mutation. (C) Upper panel, partial exon-intron organizationof bPix gene showing the point mutation effecting splicing of the gene.Lower panel, RT-PCR analysis of wild-type and bbh^(m292) mutant cDNAwith primers flanking exon-14. (D) Upper panel, the phenotypes ofbbh^(m292) mutants treated with DMSO or artesunate and imaged at 48 hpf.The arrows denote sites of hemorrhage. Lower panel, percentages ofbbh^(m292) embryos with brain hemorrhage rescued by artesunate.

FIG. 15 shows that LPS induces brain hemorrhage in developing zebrafishembryo and artemether have protective effects on LPS-induced mortality.(A) Survival curves of developing zebrafish embryos when LPS isdelivered in fish water at 24 hours post fertilization (hpf). (B) showsthat artemether in fish water had a protective effect on fish survival.(C) shows that LPS treatment of 24 hpf embryos resulted in 52% ofembryos (n=120) with brain hemorrhage (arrow points to hemorrhage). (D)Bar graph representing percent (%) cerebral hemorrhage in (C).

FIG. 16 shows that statin exacerbates LPS-induced intracerebralhemorrhage in mice. (A) Atorvastatin (50 mg/kg) treatment in addition toLPS (5 mg/kg), resulted in 100% mortality 24 hours after the treatments,while LPS treatment alone only result in 25% mortality at the same timeexamined, and statin alone did not cause any mortality (n=5). (B)Atorvastatin treatment significantly increased the number of largehemorrhages caused by LPS.

DETAILED DESCRIPTION OF THE INVENTION Glossary

The term “active” as used herein refers to the ingredient, component orconstituent of the compositions of the described invention responsiblefor the intended therapeutic effect. The term “administer” as usedherein means to give or to apply. The term “administering” as usedherein includes in vivo administration, as well as administrationdirectly to cells or a tissue ex vivo.

The term “agonist” as used herein refers to a chemical substance capableof activating a receptor to induce a full or partial pharmacologicalresponse. Receptors can be activated or inactivated by either endogenousor exogenous agonists and antagonists, resulting in stimulating orinhibiting a biological response. A physiological agonist is a substancethat creates the same bodily responses, but does not bind to the samereceptor. An endogenous agonist for a particular receptor is a compoundnaturally produced by the body which binds to and activates thatreceptor. A superagonist is a compound that is capable of producing agreater maximal response than the endogenous agonist for the targetreceptor, and thus an efficiency greater than 100%. This does notnecessarily mean that it is more potent than the endogenous agonist, butis rather a comparison of the maximum possible response that can beproduced inside a cell following receptor binding. Full agonists bindand activate a receptor, displaying full efficacy at that receptor.Partial agonists also bind and activate a given receptor, but have onlypartial efficacy at the receptor relative to a full agonist. An inverseagonist is an agent which binds to the same receptor binding-site as anagonist for that receptor and reverses constitutive activity ofreceptors. Inverse agonists exert the opposite pharmacological effect ofa receptor agonist. An irreversible agonist is a type of agonist thatbinds permanently to a receptor in such a manner that the receptor ispermanently activated. It is distinct from a mere agonist in that theassociation of an agonist to a receptor is reversible, whereas thebinding of an irreversible agonist to a receptor is believed to beirreversible. This causes the compound to produce a brief burst ofagonist activity, followed by desensitization and internalization of thereceptor, which with long-term treatment produces an effect more like anantagonist. A selective agonist is specific for one certain type ofreceptor.

The term “amplification” as used herein refers to a replication ofgenetic material that results in an increase in the number of copies ofthat genetic material.

Anatomical Terms:

When referring to animals, that typically have one end with a head andmouth, with the opposite end often having the anus and tail, the headend is referred to as the cranial end, while the tail end is referred toas the caudal end. Within the head itself, rostral refers to thedirection toward the end of the nose, and caudal is used to refer to thetail direction. The surface or side of an animal's body that is normallyoriented upwards, away from the pull of gravity, is the dorsal side; theopposite side, typically the one closest to the ground when walking onall legs, swimming or flying, is the ventral side. On the limbs or otherappendages, a point closer to the main body is “proximal”; a pointfarther away is “distal”. Three basic reference planes are used inzoological anatomy. A “sagittal” plane divides the body into left andright portions. The “midsagittal” plane is in the midline, i.e. it wouldpass through midline structures such as the spine, and all othersagittal planes are parallel to it. A “coronal” plane divides the bodyinto dorsal and ventral portions. A “transverse” plane divides the bodyinto cranial and caudal portions.

When referring to humans, the body and its parts are always describedusing the assumption that the body is standing upright. Portions of thebody which are closer to the head end are “superior” (corresponding tocranial in animals), while those farther away are “inferior”(corresponding to caudal in animals). Objects near the front of the bodyare referred to as “anterior” (corresponding to ventral in animals);those near the rear of the body are referred to as “posterior”(corresponding to dorsal in animals). A transverse, axial, or horizontalplane is an X-Y plane, parallel to the ground, which separates thesuperior/head from the inferior/feet. A coronal or frontal plane is aY-Z plane, perpendicular to the ground, which separates the anteriorfrom the posterior. A sagittal plane is an X-Z plane, perpendicular tothe ground and to the coronal plane, which separates left from right.The midsagittal plane is the specific sagittal plane that is exactly inthe middle of the body.

Structures near the midline are called medial and those near the sidesof animals are called lateral. Therefore, medial structures are closerto the midsagittal plane, lateral structures are further from themidsagittal plane. Structures in the midline of the body are median. Forexample, the tip of a human subject's nose is in the median line.

Ipsilateral means on the same side, contralateral means on the otherside and bilateral means on both sides. Structures that are close to thecenter of the body are proximal or central, while ones more distant aredistal or peripheral. For example, the hands are at the distal end ofthe arms, while the shoulders are at the proximal ends.

The term “antagonist” as used herein refers to a substance thatcounteracts the effects of another substance.

The terms “apoptosis” or “programmed cell death” refer to a highlyregulated and active process that contributes to biologic homeostasiscomprised of a series of biochemical events that lead to a variety ofmorphological changes, including blebbing, changes to the cell membrane,such as loss of membrane asymmetry and attachment, cell shrinkage,nuclear fragmentation, chromatin condensation and chromosomal DNAfragmentation, without damaging the organism.

Apoptotic cell death is induced by many different factors and involvesnumerous signaling pathways, some dependent on caspase proteases (aclass of cysteine proteases) and others that are caspase independent. Itcan be triggered by many different cellular stimuli, including cellsurface receptors, mitochondrial response to stress, and cytotoxic Tcells, resulting in activation of apoptotic signaling pathways.

The caspases involved in apoptosis convey the apoptotic signal in aproteolytic cascade, with caspases cleaving and activating othercaspases that then degrade other cellular targets that lead to celldeath. The caspases at the upper end of the cascade include caspase-8and caspase-9. Caspase-8 is the initial caspase involved in response toreceptors with a death domain (DD) like Fas.

Receptors in the tumor necrosis factor receptor family are associatedwith the induction of apoptosis, as well as inflammatory signaling. TheFas receptor (CD95) mediates apoptotic signaling by Fas-ligand expressedon the surface of other cells. The Fas-FasL interaction plays animportant role in the immune system and lack of this system leads toautoimmunity, indicating that Fas-mediated apoptosis removesself-reactive lymphocytes. Fas signaling also is involved in immunesurveillance to remove transformed cells and virus infected cells.Binding of Fas to oligomerized FasL on another cell activates apoptoticsignaling through a cytoplasmic domain termed the death domain (DD) thatinteracts with signaling adaptors including FAF, FADD and DAX toactivate the caspase proteolytic cascade. Caspase-8 and caspase-10 firstare activated to then cleave and activate downstream caspases and avariety of cellular substrates that lead to cell death.

Mitochondria participate in apoptotic signaling pathways through therelease of mitochondrial proteins into the cytoplasm. Cytochrome c, akey protein in electron transport, is released from mitochondria inresponse to apoptotic signals, and activates Apaf-1, a protease releasedfrom mitochondria. Activated Apaf-1 activates caspase-9 and the rest ofthe caspase pathway. Smac/DIABLO is released from mitochondria andinhibits IAP proteins that normally interact with caspase-9 to inhibitapoptosis. Apoptosis regulation by Bcl-2 family proteins occurs asfamily members form complexes that enter the mitochondrial membrane,regulating the release of cytochrome c and other proteins. Tumornecrosis factor family receptors that cause apoptosis directly activatethe caspase cascade, but can also activate Bid, a Bcl-2 family member,which activates mitochondria-mediated apoptosis. Bax, another Bcl-2family member, is activated by this pathway to localize to themitochondrial membrane and increase its permeability, releasingcytochrome c and other mitochondrial proteins. Bcl-2 and Bcl-xL preventpore formation, blocking apoptosis. Like cytochrome c, AIF(apoptosis-inducing factor) is a protein found in mitochondria that isreleased from mitochondria by apoptotic stimuli. While cytochrome C islinked to caspase-dependent apoptotic signaling, AIF release stimulatescaspase-independent apoptosis, moving into the nucleus where it bindsDNA. DNA binding by AIF stimulates chromatin condensation, and DNAfragmentation, perhaps through recruitment of nucleases.

The mitochondrial stress pathway begins with the release of cytochrome cfrom mitochondria, which then interacts with Apaf-1, causingself-cleavage and activation of caspase-9. Caspase-3, -6 and -7 aredownstream caspases that are activated by the upstream proteases and actthemselves to cleave cellular targets.

Granzyme B and perforin proteins released by cytotoxic T cells induceapoptosis in target cells, forming transmembrane pores, and triggeringapoptosis, perhaps through cleavage of caspases, althoughcaspase-independent mechanisms of granzyme B mediated apoptosis havebeen suggested.

Fragmentation of the nuclear genome by multiple nucleases activated byapoptotic signaling pathways to create a nucleosomal ladder is acellular response characteristic of apoptosis. One nuclease involved inapoptosis is DNA fragmentation factor (DFF), a caspase-activated DNAse(CAD). DFF/CAD is activated through cleavage of its associated inhibitorICAD by caspases proteases during apoptosis. DFF/CAD interacts withchromatin components such as topoisomerase II and histone H1 to condensechromatin structure and perhaps recruit CAD to chromatin. Anotherapoptosis activated protease is endonuclease G (EndoG). EndoG is encodedin the nuclear genome but is localized to mitochondria in normal cells.EndoG may play a role in the replication of the mitochondrial genome, aswell as in apoptosis. Apoptotic signaling causes the release of EndoGfrom mitochondria. The EndoG and DFF/CAD pathways are independent sincethe EndoG pathway still occurs in cells lacking DFF.

Hypoxia, as well as hypoxia followed by reoxygenation can triggercytochrome c release and apoptosis. Glycogen synthase kinase (GSK-3) aserine-threonine kinase ubiquitously expressed in most cell types,appears to mediate or potentiate apoptosis due to many stimuli thatactivate the mitochondrial cell death pathway (Loberg, R D, et al.(2002) J. Biol. Chem. 277 (44): 41667-673). It has been demonstrated toinduce caspase 3 activation and to activate the proapoptotic tumorsuppressor gene p53. It also has been suggested that GSK-3 promotesactivation and translocation of the proapoptotic Bcl-2 family member,Bax, which, upon aggregation and mitochondrial localization, inducescytochrome c release. Akt is a critical regulator of GSK-3, andphosphorylation and inactivation of GSK-3 may mediate some of theantiapoptotic effects of Akt.

The term “appearance” as used herein refers to an outward aspect orpresentation of oneself.

The term “apply” as used herein refers to placing in contact with or tolay or spread on.

The term “assay marker” or “reporter gene” (or “reporter”) refers to agene that can be detected, or easily identified and measured. Theexpression of the reporter gene may be measured at either the RNA level,or at the protein level. The gene product, which may be detected in anexperimental assay protocol, includes, but is not limited to, markerenzymes, antigens, amino acid sequence markers, cellular phenotypicmarkers, nucleic acid sequence markers, and the like. Researchers mayattach a reporter gene to another gene of interest in cell culture,bacteria, animals, or plants. For example, some reporters are selectablemarkers, or confer characteristics upon on organisms expressing themallowing the organism to be easily identified and assayed. To introducea reporter gene into an organism, researchers may place the reportergene and the gene of interest in the same DNA construct to be insertedinto the cell or organism. For bacteria or eukaryotic cells in culture,this may be in the form of a plasmid. Commonly used reporter genes mayinclude, but are not limited to, fluorescent proteins, luciferase,β-galactosidase, and selectable markers, such as chloramphenicol andkanomycin.

The term “associate” and its various grammatical forms as used hereinrefers to joining, connecting, or combining to, either directly,indirectly, actively, inactively, inertly, non-inertly, completely orincompletely.

The term “in association with” as used herein refers to a relationshipbetween two substances that connects, joins or links one substance withanother

The term “biomarkers” (or “biosignatures”) as used herein refers topeptides, proteins, nucleic acids, antibodies, genes, metabolites, orany other substances used as indicators of a biologic state. It is acharacteristic that is measured objectively and evaluated as a cellularor molecular indicator of normal biologic processes, pathogenicprocesses, or pharmacologic responses to a therapeutic intervention. Theterm “indicator” as used herein refers to any substance, number or ratioderived from a series of observed facts that may reveal relative changesas a function of time; or a signal, sign, mark, note or symptom that isvisible or evidence of the existence or presence thereof. Once aproposed biomarker has been validated, it may be used to diagnosedisease risk, presence of disease in an individual, or to tailortreatments for the disease in an individual (choices of drug treatmentor administration regimes). In evaluating potential drug therapies, abiomarker may be used as a surrogate for a natural endpoint, such assurvival or irreversible morbidity. If a treatment alters the biomarker,and that alteration has a direct connection to improved health, thebiomarker may serve as a surrogate endpoint for evaluating clinicalbenefit. Clinical endpoints are variables that can be used to measurehow patients feel, function or survive. Surrogate endpoints arebiomarkers that are intended to substitute for a clinical endpoint;these biomarkers are demonstrated to predict a clinical endpoint with aconfidence level acceptable to regulators and the clinical community.

The term “cDNA” refers to DNA synthesized from a mature mRNA template.cDNA most often is synthesized from mature mRNA using the enzyme reversetranscriptase. The enzyme operates on a single strand of mRNA,generating its complementary DNA based on the pairing of RNA base pairs(A, U, G, C) to their DNA complements (T, A, C, G). There are severalmethods known for generating cDNA to obtain, for example, eukaryoticcDNA whose introns have been spliced. Generally, these methodsincorporate the following steps: a) a eukaryotic cell transcribes theDNA (from genes) into RNA (pre-mRNA); b) the same cell processes thepre-mRNA strands by splicing out introns, and adding a poly-A tail and5′ methyl-guanine cap; c) this mixture of mature mRNA strands areextracted from the cell; d) a poly-T oligonucleotide primer ishybridized onto the poly-A tail of the mature mRNA template (reversetranscriptase requires this double-stranded segment as a primer to startits operation); e) reverse transcriptase is added, along withdeoxynucleotide triphosphates (A, T, G, C); f) the reverse transcriptasescans the mature mRNA and synthesizes a sequence of DNA that complementsthe mRNA template. This strand of DNA is complementary DNA (see alsoCurrent Protocols in Molecular Biology, John Wiley & Sons, incorporatedin its entirety herein).

The term “cell” is used herein to refer to the structural and functionalunit of living organisms and is the smallest unit of an organismclassified as living.

The term “cell culture” as used herein refers to establishment andmaintenance of cultures derived from dispersed cells taken from originaltissues, primary culture, or from a cell line or cell strain.

The term “cell line” as used herein refers to an immortalized cell,which have undergone transformation and can be passed indefinitely inculture.

The term “compatible” as used herein means that the components of acomposition are capable of being combined with each other in a mannersuch that there is no interaction that would substantially reduce theefficacy of the composition under ordinary use conditions.

The terms “composition” and “formulation” are used interchangeablyherein to refer to a product of the described invention that comprisesall active and inert ingredients. The terms “pharmaceutical composition”or “pharmaceutical formulation” as used herein refer to a composition orformulation that is employed to prevent, reduce in intensity, cure orotherwise treat a target condition or disease.

The term “contacting” as used herein refers to bring or put in contact,to be in or come into contact. The term “contact” as used herein refersto a state or condition of touching or of immediate or local proximity.Contacting a composition to a target destination, such as, but notlimited to, an organ, a tissue, or a cell, may occur by any means ofadministration known to the skilled artisan.

The terms “deletion” and “deletion mutation” are used interchangeablyherein to refer to that in which a base or bases are lost from the DNA.

The term “derivative” as used herein means a compound that may beproduced from another compound of similar structure in one or moresteps. A “derivative” or “derivatives” of a peptide or a compoundretains at least a degree of the desired function of the peptide orcompound. Accordingly, an alternate term for “derivative” may be“functional derivative.” Derivatives can include chemical modificationsof the peptide, such as akylation, acylation, carbamylation, iodinationor any modification that derivatizes the peptide. Such derivatizedmolecules include, for example, those molecules in which free aminogroups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,chloroacetyl groups or formal groups. Free carboxyl groups can bederivatized to form salts, esters, amides, or hydrazides. Free hydroxylgroups can be derivatized to form O-acyl or O-alkyl derivatives. Theimidazole nitrogen of histidine can be derivatized to formN-im-benzylhistidine. Also included as derivatives or analogues arethose peptides that contain one or more naturally occurring amino acidderivative of the twenty standard amino acids, for example,4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine,ornithine or carboxyglutamiate, and can include amino acids that are notlinked by peptide bonds. Such peptide derivatives can be incorporatedduring synthesis of a peptide, or a peptide can be modified bywell-known chemical modification methods (see, e.g., Glazer et al.(1975), Chemical Modification of Proteins, Selected Methods andAnalytical Procedures, Elsevier Biomedical Press, New York).

The term “detectable marker” encompasses both selectable markers andassay markers. The term “selectable markers” refers to a variety of geneproducts to which cells transformed with an expression construct can beselected or screened, including drug-resistance markers, antigenicmarkers useful in fluorescence-activated cell sorting, adherence markerssuch as receptors for adherence ligands allowing selective adherence,and the like. When a nucleic acid is prepared or altered synthetically,advantage can be taken of known codon preferences of the intended hostwhere the nucleic acid is to be expressed.

The term “detectable response” refers to any signal or response that maybe detected in an assay, which may be performed with or without adetection reagent. Detectable responses include, but are not limited to,radioactive decay and energy (e.g., fluorescent, ultraviolet, infrared,visible) emission, absorption, polarization, fluorescence,phosphorescence, transmission, reflection or resonance transfer.Detectable responses also include chromatographic mobility, turbidity,electrophoretic mobility, mass spectrum, ultraviolet spectrum, infraredspectrum, nuclear magnetic resonance spectrum and x-ray diffraction.Alternatively, a detectable response may be the result of an assay tomeasure one or more properties of a biologic material, such as meltingpoint, density, conductivity, surface acoustic waves, catalytic activityor elemental composition. A “detection reagent” is any molecule thatgenerates a detectable response indicative of the presence or absence ofa substance of interest. Detection reagents include any of a variety ofmolecules, such as antibodies, nucleic acid sequences and enzymes. Tofacilitate detection, a detection reagent may comprise a marker.

The term “differentiation” as used herein refers to a property of cellsto exhibit tissue-specific differentiated properties in culture.

The term “effective amount” refers to the amount necessary or sufficientto realize a desired biologic effect.

The term “EC50” as used herein refers to the concentration (expressed inmolar units or g/L) of a drug that produces 50% of the maximal possibleeffect of that drug.

The term “expression system” refers to a genetic sequence, whichincludes a protein encoding region operably linked to all of the geneticsignals necessary to achieve expression of the protein encoding region.Traditionally, the expression system will include a regulatory elementsuch as, for example, a promoter or enhancer, to increase transcriptionand/or translation of the protein encoding region, or to provide controlover expression. The regulatory element may be located upstream ordownstream of the protein encoding region, or may be located at anintron (non-coding portion) interrupting the protein encoding region.Alternatively, it also is possible for the sequence of the proteinencoding region itself to comprise regulatory ability.

The term “hpf” as used herein refers to hours post fertilization.

The term “hybridization” refers to the process of combiningcomplementary, single-stranded nucleic acids into a single molecule.Nucleotides will bind to their complement under normal conditions, sotwo perfectly complementary strands will bind (or ‘anneal’) to eachother readily. However, due to the different molecular geometries of thenucleotides, a single inconsistency between the two strands will makebinding between them more energetically unfavorable. Measuring theeffects of base incompatibility by quantifying the rate at which twostrands anneal can provide information as to the similarity in basesequence between the two strands being annealed. The term “specificallyhybridizes” as used herein refers to the process whereby a nucleic aciddistinctively or definitively forming base pairs with complementaryregions of at least one strand of DNA that was not originally paired tothe nucleic acid. A nucleic acid that selectively hybridizes undergoeshybridization, under stringent hybridization conditions, of the nucleicacid sequence to a specified nucleic acid target sequence to adetectably greater degree (e.g., at least 2-fold over background) thanits hybridization to non-target nucleic acid sequences and to thesubstantial exclusion of non-target nucleic acids. Selectivelyhybridizing sequences typically have about at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity, or 100%sequence identity (i.e., complementary) with each other.

The term “hypomorphic mutation” as used herein refers to a type ofmutation in which the altered gene product possesses a reduced level ofactivity, or in which the wild-type gene product is expressed at areduced level.

The terms “inhibiting”, “inhibit” or “inhibition” are used herein torefer to reducing the amount or rate of a process, to stopping theprocess entirely, or to decreasing, limiting, or blocking the action orfunction thereof. Inhibition may include a reduction or decrease of theamount, rate, action function, or process of a substance by at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99%.

The term “inhibitor” as used herein refers to a molecule that binds toan enzyme thereby decreasing enzyme activity. Enzyme inhibitors aremolecules that bind to enzymes thereby decreasing enzyme activity. Thebinding of an inhibitor may stop substrate from entering the active siteof the enzyme and/or hinder the enzyme from catalyzing its reaction.Inhibitor binding is either reversible or irreversible. Irreversibleinhibitors usually react with the enzyme and change it chemically, forexample, by modifying key amino acid residues needed for enzymaticactivity. In contrast, reversible inhibitors bind non-covalently andproduce different types of inhibition depending on whether theseinhibitors bind the enzyme, the enzyme-substrate complex, or both.Enzyme inhibitors often are evaluated by their specificity and potency.

An “isolated molecule” is a molecule that is substantially pure and isfree of other substances with which it is ordinarily found in nature orin vivo systems to an extent practical and appropriate for its intendeduse. In particular, the compositions are sufficiently pure and aresufficiently free from other biological constituents of host cells so asto be useful in, for example, producing pharmaceutical preparations orsequencing if the composition is a nucleic acid, peptide, orpolysaccharide. Because compositions may be admixed with apharmaceutically-acceptable carrier in a pharmaceutical preparation, thecompositions may comprise only a small percentage by weight of thepreparation. The composition is nonetheless substantially pure in thatit has been substantially separated from the substances with which itmay be associated in living systems or during synthesis. As used herein,the term “substantially pure” refers purity of at least 75%, at least80%, at least 85%, at least 90%, at least 95% or at least 99% pure asdetermined by an analytical protocol. Such protocols may include, forexample, but are not limited to, fluorescence activated cell sorting,high performance liquid chromatography, gel electrophoresis,chromatography, and the like.

The term “minimizing progression” as used herein refers to reducing theamount, extent, size, or degree of development of a sequence or seriesof events.

The term “modulate” as used herein means to regulate, alter, adapt, oradjust to a certain measure or proportion.

The term “morpholino oligonucleotides (MO)” as used herein refer tononionic DNA analogs with a phosphorodiamidate molecular backbone, whichblocks access of other molecules to specific sequences within antisensenucleic acid sequences. Although they possess altered backbone linkagescompared with DNA or RNA, morpholinos bind to complementary nucleic acidsequences by Watson-Crick base-pairing. This binding is no tighter thanbinding of analogous DNA and RNA oligomers, necessitating the use ofrelatively long 25-base morpholinos for antisense gene inhibition. Thebackbone makes morpholinos resistant to digestion by nucleases. Also,because the backbone lacks negative charge, it is thought thatmorpholinos are less likely to interact nonselectively with cellularproteins; such interactions often obscure the observation of informativephenotypes (Corey, D. R. and J. M. Abrams (2001) “Morpholino antisenseoligonucleotides: tools for investigating vertebrate development,”Genome Biol. 2(5): 1015.1-1015.3). Duplex formation between MOs and mRNAprevents translation through MO hybridization near the mRNA translationinitiation codon and disrupts correct splicing by targeting the splicedonor site Wada, T. et al (2012) “Antisense morpholino targeting justupstream from a poly(A) tail junction of material mRNA removes the tailand inhibits translation,” Nucleic Acids Res. 40 (22): e173).

The term “mutation” as used herein refers to a change of the DNAsequence within a gene or chromosome of an organism resulting in thecreation of a new character or trait not found in the parental type, orthe process by which such a change occurs in a chromosome, eitherthrough an alteration in the nucleotide sequence of the DNA coding for agene or through a change in the physical arrangement of a chromosome.Three mechanisms of mutation include substitution (exchange of one basepair for another), addition (the insertion of one or more bases into asequence), and deletion (loss of one or more base pairs).

The term “nucleic acid” is used herein to refer to a DNA or RNA polymerin either single- or double-stranded form, and unless otherwise limited,encompasses known analogues having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to naturally occurring nucleotides (e.g., MOoligonucleotides).

The term “nucleotide” is used herein to refer to a chemical compoundthat consists of a heterocyclic base, a sugar, and one or more phosphategroups. In the most common nucleotides, the base is a derivative ofpurine or pyrimidine, and the sugar is the pentose deoxyribose orribose. Nucleotides are the monomers of nucleic acids, with three ormore bonding together in order to form a nucleic acid. Nucleotides arethe structural units of RNA, DNA, and several cofactors, including, butnot limited to, CoA, FAD, DMN, NAD, and NADP. Purines include adenine(A), and guanine (G); pyrimidines include cytosine (C), thymine (T), anduracil (U).

The phrase “operably linked” refers to a first sequence(s) or domainbeing positioned sufficiently proximal to a second sequence(s) or domainso that the first sequence(s) or domain can exert influence over thesecond sequence(s) or domain or a region under control of that secondsequence or domain.

The term “polynucleotide” refers to a DNA, RNA or analogs thereof thathave the essential nature of a natural ribonucleotide in that theyhybridize, under stringent hybridization conditions, to substantiallythe same nucleotide sequence as naturally occurring nucleotides and/orallow translation into the same amino acid(s) as the naturally occurringnucleotide(s). A polynucleotide may be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including among other things,simple and complex cells.

The term “pharmaceutical composition” as used herein refers to acomposition that is employed to prevent, reduce in intensity, cure orotherwise treat a target condition, syndrome, disorder or disease.

The term “pharmaceutically acceptable carrier” as used herein refers toany substantially non-toxic carrier conventionally useable foradministration of pharmaceuticals in which the isolated polypeptide ofthe present invention will remain stable and bioavailable. Thepharmaceutically acceptable carrier must be of sufficiently high purityand of sufficiently low toxicity to render it suitable foradministration to the mammal being treated. It further should maintainthe stability and bioavailability of an active agent. Thepharmaceutically acceptable carrier can be liquid or solid and isselected, with the planned manner of administration in mind, to providefor the desired bulk, consistency, etc., when combined with an activeagent and other components of a given composition.

The term “pharmaceutically acceptable salt” as used herein refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. When used inmedicine the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof. Such salts include,but are not limited to, those prepared from the following acids:hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic,acetic, salicylic, p-toluene sulphonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, andbenzene sulphonic. Also, such salts may be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts of thecarboxylic acid group. By “pharmaceutically acceptable salt” is meantthose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well-known in the art. For example, P. H. Stahl, etal. describe pharmaceutically acceptable salts in detail in “Handbook ofPharmaceutical Salts: Properties, Selection, and Use” (Wiley VCH,Zurich, Switzerland: 2002). The salts may be prepared in situ during thefinal isolation and purification of the compounds described within thepresent invention or separately by reacting a free base function with asuitable organic acid. Representative acid addition salts include, butare not limited to, acetate, adipate, alginate, citrate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,camphorsulfonate, digluconate, glycerophosphate, hemisulfate,heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethansulfonate(isethionate), lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate and undecanoate. Also, the basicnitrogen-containing groups may be quaternized with such agents as loweralkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyland diamyl sulfates; long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides; arylalkyl halides likebenzyl and phenethyl bromides and others. Water or oil-soluble ordispersible products are thereby obtained. Examples of acids which maybe employed to form pharmaceutically acceptable acid addition saltsinclude such inorganic acids as hydrochloric acid, hydrobromic acid,sulphuric acid and phosphoric acid and such organic acids as oxalicacid, maleic acid, succinic acid and citric acid. Basic addition saltsmay be prepared in situ during the final isolation and purification ofcompounds described within the invention by reacting a carboxylicacid-containing moiety with a suitable base such as the hydroxide,carbonate or bicarbonate of a pharmaceutically acceptable metal cationor with ammonia or an organic primary, secondary or tertiary amine.Pharmaceutically acceptable salts include, but are not limited to,cations based on alkali metals or alkaline earth metals such as lithium,sodium, potassium, calcium, magnesium and aluminum salts and the likeand nontoxic quaternary ammonia and amine cations including ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, diethylamine, ethylamine and the like.Other representative organic amines useful for the formation of baseaddition salts include ethylenediamine, ethanolamine, diethanolamine,piperidine, piperazine and the like. Pharmaceutically acceptable saltsalso may be obtained using standard procedures well known in the art,for example, by reacting a sufficiently basic compound such as an aminewith a suitable acid affording a physiologically acceptable anion.Alkali metal (for example, sodium, potassium or lithium) or alkalineearth metal (for example calcium or magnesium) salts of carboxylic acidsmay also be made.

The term “primer” refers to a nucleic acid which, when hybridized to astrand of DNA, is capable of initiating the synthesis of an extensionproduct in the presence of a suitable polymerization agent. The primeris sufficiently long to uniquely hybridize to a specific region of theDNA strand. A primer also may be used on RNA, for example, to synthesizethe first strand of cDNA.

The term “promoter” refers to a region of DNA upstream, downstream, ordistal, from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.For example, T7, T3 and Sp6 are RNA polymerase promoter sequences. InRNA synthesis, promoters are a means to demarcate which genes should beused for messenger RNA creation and by extension, control which proteinsthe cell manufactures. Promoters represent critical elements that canwork in concert with other regulatory regions (enhancers, silencers,boundary elements/insulators) to direct the level of transcription of agiven gene.

The term “reduced” or “to reduce” as used herein refer to adiminishment, a decrease, an attenuation or abatement of the degree,intensity, extent, size, amount, density or number of.

The term “refractory” as used herein refers to the state of beingunaffected, unresponsive, resistant or not fully responsive.

The term “restriction digestion” refers to a procedure used to prepareDNA for analysis or other processing. Also known as DNA fragmentation,it uses a restriction enzyme to selectively cleave strands of DNA intoshorter segments.

The term “restriction enzyme” (or restriction endonuclease) refers to anenzyme that cuts double-stranded DNA.

The term “restriction sites” or “restriction recognition sites” refer toparticular sequences of nucleotides that are recognized by restrictionenzymes as sites to cut a DNA molecule. The sites are generally, but notnecessarily, palindromic, (because restriction enzymes usually bind ashomodimers) and a particular enzyme may cut between two nucleotideswithin its recognition site, or somewhere nearby.

The term “Rho” as used herein refers to a subfamily of proteins relatedto the RAS subgroup thought to be involved in cell transformation andthe regulation of morphology and function of dendritic cells.Non-limiting examples of Rho proteins include RhoA, RhoB and RhoC, RhoG,RhoH, RhoQ, RhoU RhoV, Rnd1, 2 and 3 (e.g., RhoE), and RAC1, 2, 3 and 4.

Sequence:

The following terms are used herein to describe the sequencerelationships between two or more nucleic acids or polynucleotides: (a)“reference sequence”, (b) “comparison window”, (c) “sequence identity”,(d) “percentage of sequence identity”, and (e) “substantial identity”.

The term “reference sequence” refers to a sequence used as a basis forsequence comparison. A reference sequence may be a subset or theentirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

The term “comparison window” refers to a contiguous and specifiedsegment of a polynucleotide sequence, wherein the polynucleotidesequence may be compared to a reference sequence and wherein the portionof the polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be at least 30contiguous nucleotides in length, at least 40 contiguous nucleotides inlength, at least 50 contiguous nucleotides in length, at least 100contiguous nucleotides in length, or longer. Those of skill in the artunderstand that to avoid a high similarity to a reference sequence dueto inclusion of gaps in the polynucleotide sequence, a gap penaltytypically is introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman (1981), Adv. Appl.Math. 2:482; by the homology alignment algorithm of Needleman and Wunsch(1970), J. Mol. Biol. 48:443; by the search for similarity method ofPearson and Lipman (1988), Proc. Natl. Acad. Sci. 85:2444; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis., USA; the CLUSTAL program is well described byHiggins and Sharp (1988), Gene 73:237-244; Higgins and Sharp (1989)CABIOS 5:151-153; Corpet, et al. (1988) Nucleic Acids Research16:10881-90; Huang, et al. (1992) Computer Applications in theBiosciences 8:155-65, and Pearson, et al. (1994) Methods in MolecularBiology 24:307-331. The BLAST family of programs, which can be used fordatabase similarity searches, includes: BLASTN for nucleotide querysequences against nucleotide database sequences; BLASTX for nucleotidequery sequences against protein database sequences; BLASTP for proteinquery sequences against protein database sequences; TBLASTN for proteinquery sequences against nucleotide database sequences; and TBLASTX fornucleotide query sequences against nucleotide database sequences. See,Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995).

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the BLAST 2.0 suite of programsusing default parameters (Altschul et al. (1997) Nucleic Acids Res.25:3389-3402). Software for performing BLAST analyses is publiclyavailable, e.g., through the National Center forBiotechnology-Information (http://www.hcbi.nlm.nih.gov/). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits then are extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always>0) and N (penalty score formismatching residues; always<0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a word length (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. BLAST searches assume thatproteins may be modeled as random sequences. However, many real proteinscomprise regions of nonrandom sequences which may be homopolymerictracts, short-period repeats, or regions enriched in one or more aminoacids. Such low-complexity regions may be aligned between unrelatedproteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs may be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen (1993), Comput. Chem., 17:149-163) and XNU (Claverie andStates (1993) Comput. Chem., 17:191-201) low-complexity filters may beemployed alone or in combination.

The term “sequence identity” or “identity” in the context of two nucleicacid or polypeptide sequences is used herein to refer to the residues inthe two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions that are not identical often differ by conservativeamino acid substitutions, i.e., where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g. charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity”. Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller (1988) Computer Applic. Biol. Sci.,4:11-17, e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

The term “percentage of sequence identity” as used herein means thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 70% sequenceidentity, at least 80% sequence identity, at least 90% sequence identityand at least 95% sequence identity, compared to a reference sequenceusing one of the alignment programs described using standard parameters.One of skill will recognize that these values may be adjustedappropriately to determine corresponding identity of proteins encoded bytwo nucleotide sequences by taking into account codon degeneracy, aminoacid similarity, reading frame positioning and the like. Substantialidentity of amino acid sequences for these purposes normally meanssequence identity of at least 60%, or at least 70%, at least 80%, atleast 90%, or at least 95%. Another indication that nucleotide sequencesare substantially identical is if two molecules hybridize to each otherunder stringent conditions. However, nucleic acids that do not hybridizeto each other under stringent conditions are still substantiallyidentical if the polypeptides that they encode are substantiallyidentical. This may occur, e.g., when a copy of a nucleic acid iscreated using the maximum codon degeneracy permitted by the geneticcode. One indication that two nucleic acid sequences are substantiallyidentical is that the polypeptide that the first nucleic acid encodes isimmunologically cross reactive with the polypeptide encoded by thesecond nucleic acid.

The terms “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with at least 70% sequence identityto a reference sequence, at least 80%, at least 85%, at least 90% or 95%sequence identity to the reference sequence over a specified comparisonwindow. Optionally, optimal alignment is conducted using the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443.An indication that two peptide sequences are substantially identical isthat one peptide is immunologically reactive with antibodies raisedagainst the second peptide. Thus, a peptide is substantially identicalto a second peptide, for example, where the two peptides differ only bya conservative substitution. Peptides which are “substantially similar”share sequences as noted above except that residue positions that arenot identical may differ by conservative amino acid changes.

The term “subject” or “individual” or “patient” are used interchangeablyto refer to a member of an animal species of vertebrate origin, e.g., azebrafish, to mammalian origin, including but not limited to, mouse,rat, cat, goat, sheep, horse, hamster, ferret, pig, dog, platypus,guinea pig, rabbit and a primate, such as, for example, a monkey, ape,or human.

The phrase “subject in need thereof” as used herein refers to a patientthat (i) susceptible to ICH, BMH or CCM that will be administered atherapeutic agent according to the described invention to treat the ICH,BMH or CCM, (ii) is receiving a therapeutic agent according to thedescribed invention to treat ICH/BMH or CCM; or (iii) has received atherapeutic agent according to the described invention to treat ICH/BMHor CCM, unless the context and usage of the phrase indicates otherwise.

The term “substitution” is used herein to refer to that in which a baseor bases are exchanged for another base or bases in DNA. Substitutionsmay be synonymous substitutions or nonsynonymous substitutions. As usedherein, “synonymous substitutions” refer to substitutions of one basefor another in an exon of a gene coding for a protein, such that theamino acid sequence produced is not modified. The term “nonsynonymoussubstitutions” as used herein refer to substitutions of one base foranother in an exon of a gene coding for a protein, such that the aminoacid sequence produced is modified.

The term “susceptible” as used herein refers to a member of a populationat risk.

The term “therapeutic agent” as used herein refers to a drug, molecule,nucleic acid, protein, composition or other substance that provides atherapeutic effect. The term “active” as used herein refers to theingredient, component or constituent of the compositions of the presentinvention responsible for the intended therapeutic effect. The terms“therapeutic agent” and “active agent” are used interchangeably herein.The active agent may be a therapeutically effective amount of at leastone of an active agent itself, a mimic, a derivative, an agonist of thatactive agent, or a pharmaceutically acceptable salt thereof.

The term “therapeutic component” as used herein refers to atherapeutically effective dosage (i.e., dose and frequency ofadministration) that eliminates, reduces, or prevents the progression ofa particular disease manifestation in a percentage of a population. Anexample of a commonly used therapeutic component is the ED₅₀, whichdescribes the dose in a particular dosage that is therapeuticallyeffective for a particular disease manifestation in 50% of a population.

The term “therapeutic effect” as used herein refers to a consequence oftreatment, the results of which are judged to be desirable andbeneficial. A therapeutic effect may include, directly or indirectly,the arrest, reduction, or elimination of a disease manifestation. Atherapeutic effect also may include, directly or indirectly, the arrestreduction or elimination of the progression of a disease manifestation.

The terms “therapeutic amount”, an “amount effective”, or“pharmaceutical amount” of one or more of the active agents and usedinterchangeably to refer to an amount that is sufficient to provide theintended benefit of treatment.

The intensity of effect of a drug (y-axis) can be plotted as a functionof the dose of drug administered (X-axis) (Goodman & Gilman's ThePharmacological Basis of Therapeutics, Ed. Joel G. Hardman, Lee E.Limbird, Eds., 10th Ed., McGraw Hill, New York (2001), p. 25, 50). Theseplots are referred to as dose-effect curves. Such a curve can beresolved into simpler curves for each of its components. Theseconcentration-effect relationships can be viewed as having fourcharacteristic variables: potency, slope, maximal efficacy, andindividual variation.

The location of the dose-effect curve along the concentration axis is anexpression of the potency of a drug (Id).

The slope of the dose-effect curve reflects the mechanism of action of adrug. The steepness of the curve dictates the range of doses useful forachieving a clinical effect.

The terms “maximal efficacy” or “clinical efficacy” as usedinterchangeably herein refer to the maximal effect that can be producedby a drug. Maximal efficacy is determined principally by the propertiesof the drug and its receptor-effector system and is reflected in theplateau of the curve. In clinical use, a drug's dosage may be limited byundesired effects.

The term “biological variability” as used herein refers to an effect ofvarying intensity that may occur in different individuals at a specifiedconcentration of a drug. It follows that a range of concentrations maybe required to produce an effect of specified intensity in all subjects.

Lastly, different individuals may vary in the magnitude of theirresponse to the same concentration of a drug when the appropriatecorrection has been made for differences in potency, maximal efficacyand slope.

The duration of a drug's action is determined by the time period overwhich concentrations exceed the minimum effective concentration (MEC).Following administration of a dose of drug, its effects usually show acharacteristic temporal pattern. A plot of drug effect versus timeillustrates the temporal characteristics of drug effect and itsrelationship to the therapeutic window. A lag period is present beforethe drug concentration exceeds the MEC for the desired effect. Followingonset of the response, the intensity of the effect increases as the drugcontinues to be absorbed and distributed. This reaches a peak, afterwhich drug elimination results in a decline in the effect's intensitythat disappears when the drug concentration falls back below the MEC.The therapeutic window reflects a concentration range that providesefficacy without unacceptable toxicity. Accordingly another dose of drugshould be given to maintain concentrations within the therapeuticwindow.

The term “transcription termination signal” refers to a section ofgenetic sequence that marks the end of gene or operon on genomic DNA fortranscription. In prokaryotes, two classes of transcription terminationsignals are known: 1) intrinsic transcription termination signals wherea hairpin structure forms within the nascent transcript that disruptsthe mRNA-DNA-RNA polymerase ternary complex; and 2) Rho-dependenttranscription termination signal that require Rho factor, an RNAhelicase protein complex to disrupt the nascent mRNA-DNA-RNA polymeraseternary complex. In eukaryotes, transcription termination signals arerecognized by protein factors that co-transcriptionally cleave thenascent RNA at a polyadenylation signal (i.e, “poly-A signal” or “poly-Atail”) halting further elongation of the transcript by RNA polymerase.The subsequent addition of the poly-A tail at this site stabilizes themRNA and allows it to be exported outside the nucleus. Terminationsequences are distinct from termination codons that occur in the mRNAand are the stopping signal for translation, which also may be callednonsense codons.

The term “treat” or “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a disease, conditionor disorder, substantially ameliorating clinical or esthetical symptomsof a condition, substantially preventing the appearance of clinical oresthetical symptoms of a disease, condition, or disorder, and protectingfrom harmful or annoying symptoms. Treating further refers toaccomplishing one or more of the following: (a) reducing the severity ofthe disorder; (b) limiting development of symptoms characteristic of thedisorder(s) being treated; (c) limiting worsening of symptomscharacteristic of the disorder(s) being treated; (d) limiting recurrenceof the disorder(s) in patients that have previously had the disorder(s);and (e) limiting recurrence of symptoms in patients that were previouslyasymptomatic for the disorder(s).

The terms “variants”, “mutants”, and “derivatives” are used herein torefer to nucleotide sequences with substantial identity to a referencenucleotide sequence. The differences in the sequences may by the resultof changes, either naturally or by design, in sequence or structure.Natural changes may arise during the course of normal replication orduplication in nature of the particular nucleic acid sequence. Designedchanges may be specifically designed and introduced into the sequencefor specific purposes. Such specific changes may be made in vitro usinga variety of mutagenesis techniques. Such sequence variants generatedspecifically may be referred to as “mutants” or “derivatives” of theoriginal sequence.

The term “vascular leakage” as used herein refers to a pathologicincrease in vascular permeability.

The term “vascular permeability” as used herein refers to the net amountof a solute, typically a macromolecule that has crossed a vascular bedand accumulated in the interstitium in response to a vascularpermeabilizing agent or at a site of pathological angiogenesis.

The term “vascular stability” as used herein includes the control ofendothelial cell cytoskeleton and junction proteins and the interactionof endothelial cells with mural cells.

The term “wild-type” as used herein refers to the typical form of anorganism, strain, gene, protein, nucleic acid, or characteristic as itoccurs in nature. Wild-type refers to the most common phenotype in thenatural population. The terms “wild-type” and “naturally occurring” areused interchangeably.

According to one aspect, the described invention provides a method forreducing incidence of bleeding in the brain by administering apharmaceutical composition containing a small molecule therapeuticcompound, a therapeutic amount of which is effective to reduce incidenceof bleeding in the brain by at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, by 60% or less, by 55% or less,by 50% or less, by 45% or less, by 40% or less, by 35% or less, by 30%or less, relative to a control.

According to some embodiments, the small molecule therapeutic compoundis selected from the group consisting of artemether or a derivative ofartemether. According to some embodiments, the derivative of artemisininis dihydroartemisinin, artemisinin, or artesunate.

According to some embodiments, the small molecule therapeutic compoundis selected from the group consisting of benidipine, lacidipine,ethynylestradiol or triptolide.

According to some embodiments, the bleeding in the brain is induced by astatin, by a lipopolysaccharide, or both.

According to some embodiments, the statin is atorvastatin.

According to some embodiments the bleeding in the brain is a spontaneousintracerebral hemorrhage.

According to some embodiments, the spontaneous intracerebral hemorrhageoccurs in association with administration of a statin.

According to some embodiments, the bleeding in the brain is a brainmicrohemorrhage.

According to some embodiments, the brain microhemorrhage occurs inassociation with administration of a statin.

According to some embodiments, the bleeding in the brain comprises abrain vascular malformation.

According to some embodiments, the brain vascular malformation is acerebral cavernous malformation.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, exemplarymethods and materials have been described. All publications mentionedherein are incorporated herein by reference to disclose and describedthe methods and/or materials in connection with which the publicationsare cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural references unlessthe context clearly dictates otherwise.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application and eachis incorporated by reference in its entirety. Nothing herein is to beconstrued as an admission that the present invention is not entitled toantedate such publication by virtue of prior invention. Further, thedates of publication provided may be different from the actualpublication dates which may need to be independently confirmed.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Materials and Methods

Zebrafish Husbandry

All zebrafish (Danio rerio) experiments were conducted under St.Michael's Hospital Animal Care Committee (Toronto, Ontario, Canada)approved protocol ACC403. The zebrafish were housed in the Li Ka ShingKnowledge Institute (St. Michael's Hospital, Toronto, Ontario, Canada)research vivarium and maintained and staged as previously described(Avdesh A, Chen M, Martin-Iverson M T et al. Regular care andmaintenance of a zebrafish (Danio rerio) laboratory: an introduction. JVis Exp 2012;e4196). In short, the fish were housed under a 14 hlight:10 h dark cycle at 28° C. Embryos were produced by pair mating andraised in 1×E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂,0.33 mM MgSO₄. Strains used in this study included Tg(Flk:GFP;Gata:dsRed) and bbh(m292); kdrl:mCherry −/−). The collection offertilized eggs was obtained through pair-wise breeding according to thestandard method previously described (Id.).

Statin-Induced Brain Hemorrhage in Zebrafish

Zebrafish as a model for hemorrhagic stroke has been proposed previously(Butler M G, Gore A V, Weinstein B M. Zebrafish as a model forhemorrhagic stroke. Methods Cell Biol 2011; 105:137-161). In addition togenetic models of brain hemorrhage, statins have been used to inducebrain hemorrhage in zebrafish (Gjini E, Hekking L H, Kuchler A et al.Zebrafish Tie-2 shares a redundant role with Tie-1 in heart developmentand regulates vessel integrity. Dis Model Mech 2011; 4:57-66; Eisa-BeygiS, Hatch G, Noble S, Ekker M, Moon T W. The3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) pathway regulatesdevelopmental cerebral-vascular stability via prenylation-dependentsignalling pathway. Dev Biol 2013; 373:258-266). A statin-induced modelwas adopted for our NIH drug library screening project.

Zebrafish were set up the night before the experiment day. In themorning of the experimental day, we put breeders together for mating andfertilization. 6 hours postfertilization (hpf), statins was added into a96-well plate holding 100 μl water with 7 to 8 fish eggs in each well,which was optimized through a serial pilot experiments. Statins weredissolved in DMSO and diluted with 0.5% of DMSO water into a workingsolution. 100 μl water containing 0.5% of DMSO was the medium for allwells in the final assessment.

Initially, we tested both simvastatin and atorvastatin for induction ofbrain hemorrhage. Simvastatin was tested in final concentrations of 10,25, 50, 100, and 200 nmol/L, and atorvastatin (ATV) was tested inconcentrations of 50, 150, 300, 500 nmol/L and 1 μM. After severalbatches of experiments, we found that ATV at 1 μM gave the bestreproducible brain hemorrhage in more than 80% of the larvae fish.Therefore, all subsequent screening work was done with 1 μM ATV toinduce brain hemorrhage in larvae zebrafish. Simvastatin (MW 558.6) waspurchased from Cayman Chemical (Ann Arbor, Mich.) and atorvastatincalcium salt (MW 604.69) was purchased from Sigma (St Louis, Mo.).

For screening NIH compound libraries, 5 μM of each of the drugs from thelibrary was added at 24 hpf into wells containing fish eggs treated with1 μM ATV since 6 hpf. Hemorrhage positive control wells were treatedwith ATV but not treated with any drugs. Negative controls were nottreated with any chemicals (fish with 0.5% of DMSO water).Geranylgeranyl pyrophosphate (GGPP, 4 mg/L) was used as positive rescuecontrol.

Brain hemorrhage was assessed 72 hpf (66 hours after addition ofstatins) using stereomicroscopy by two observers. Percentage of brainhemorrhage was used as final readout. Compounds showing more than 70% ofrescue of the brain hemorrhages in the initial test were re-tested togenerate a final list of hits from the library.

Four other compounds plus artesunate and artemether were independentlyidentified as positive hits from the library. Their derivatives(artemisinin and dihydro-artemisinin) were acquired (Sequoia ResearchProducts, Pangbourne, UK) and tested positive in the same ATV zebrafishmodel. All subsequent EC50 assays of positive compounds were performedwith protocols established and optimized during the screening.

Morpholino Injection

Morpholino oligonucleotides (MOs) were custom-synthesized by Gene Tools(Carvalis, Oreg.); their sequences are shown in Table 1.

TABLE 1 Morpholino sequences SEQ ID Morpholino Sequence NO: Rap1bEx35′-AAATGATGCAGAACTT SEQ ID GCCTTTCTG-3′ NO: 1 cdh5exon25′-TACAAGACCGTCTACC SEQ ID TTTCCAATC-3′ NO: 2 βPixexon65′-GCGCATCTCTCTTACC SEQ ID ACATTATAG-3′ NO: 3 pak2aexon85′-AATAGAGTACAACATA SEQ ID CCTCTTGGC-3′ NO: 4 Hmgcrb-5′-AACTGCATTCATAAAC SEQ ID splice TCACCCAGT-3′ NO: 5 Pggtl-MO/5′-CACGCGGTGTGTGGAC SEQ ID ggtasel TCACGGTCA-3′ NO: 6 splice Liss Std5′-CCTCTTACCTCAGTTA SEQ ID Control CAATTTATA-3′ NO: 7

Danieau buffer (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO₄, 0.6 mM Ca(NO₃)₂,5.0 mM HEPES, pH 7.6) was used to dilute the MO solutions to 0.2 mMfinal concentration. Individual wells were placed on a 1.0% agaroseplate, in which the embryos were positioned. Afterwards, the MO solutionwas injected through the cell yolk into embryos of 1 to 4 cell-stage.The injected quantities varied from 0.5 to 15 ng.

bbhm292 zebrafish mutant has a hypomorphic mutation in βPix, resultingin ICH/BMH and hydrocephalus. The MO is βPixexon6-MO which blockssplicing of exon 6 and results in premature protein termination.Injection of 0.2 ng of βPixexon6-MO resulted in ICH in 61% of embryos.Higher doses of βPixexon6-MO (up to 8 ng) result in a lack of bloodcirculation, and therefore no ICH/BMH was detected. Injection of 8 ngresults in complete missplicing of βPix and therefore a null phenotype,whereas lower doses retain some normally spliced βPix. The βPixexon6-MOsequence is 5′-GCGCATCTCTCTTACCACATTATAG-3′ [SEQ ID NO: 1]. βPixexon6-MOwas injected into the embryos at the 1-2 cell stage, and compounds wereadded 12 hpf. Artesunate, 5 μmol/L, prevented ICH (FIG. 14).

Another MO was designed to block the splice-donor sites after exon 8 inpak2a. Pak2a is the gene mutated in the rhdmi149 zebrafish mutant thatdevelops ICH/BMH. The pak2a-MO sequence is5′-AATAGAGTACAACATACCTCTTGGC-3′ (SEQ ID NO: 2). Eight pg of pak2a-MO wasinjected per embryo, resulting in −80% of embryos with ICH/BMH with lowmortality (FIGS. 12 and 14).

FIG. 12 depicts the pharmacological and genetic induction of loss ofcerebrovascular stabilization in developing zebrafish. (A) The putativerelationship between the HMGCR (hmgcrb)-mediated metabolic pathway andRho GTPase (CDC42) signalling in zebrafish is shown. The process ofgeranylgeranylation, catalysed by GGTase I (pggt1b), facilitatestranslocation of CDC42 to the plasma membrane. The membrane-bound CDC42functions as a molecular switch by alternating between a GDP-bound(inactive) state and a GTP-bound (active) state. βpix is a guanineexchange factor (GEF), as it activates CDC42 by stimulating GDP releaseand increasing enzyme affinity for GTP. The p21-activated kinase 2a(pak2a) is a binding partner for βPix. Pak2a is serine/threonine kinaseacting downstream of Rho GTPase signalling and are involved in thetransduction of this pathway. HMGCR function was inhibited using asplice inducing anti-sense morpholino oligonucleotide (MO) orwater-borne exposure of embryos to statins (0.5 mg/L). The functions ofpggt1b, βpix, or pak2a were reduced using gene-specific MOs. (B)VE-cadherin-mediated cell-cell adhesion is regulated in part by CDC42.When CDC42 is prenylated and in its GTP-bound active form, it interactswith the α and β-catenins to maintain the VE-cadherin-catenin complex,hence conferring stability. (C) By contrast, the unprenylated CDC42remains inactive (GDP-bound) and associated with guanine nucleotidedissociation inhibitor (GDI). This condition confers the weak adhesiveactivity, hence disrupted cell-cell stability. (D-H) Loss of the hmgcrb,pggt1b, βpix or pak2a genes precipitate cerebral hemorrhages. Ascompared with un-injected embryos in (D), those injected with MOstargeting hmgcrb (E), pggt1b (F), βpix (G), or pak2a (H) exhibited ICHphenotype at 36-52 hpf. Arrows denote the sites of abnormal accumulationof blood. Representative images are shown. Anterior is to the left.(I-L) Hemorrhages arise due to vascular defects in the brain. (I and J)Representative photomicrographs of Tg(fli1:EGFP);(gata-1:DsRed) embryosincubated in DMSO or 0.5 mg/L atorvastatin at 2 hpf and imaged at 36hpf. The arrows in (J) indicate areas where stagnant DsRed-positiveerythrocyte accumulation is observed. The arrows in (L) denote theunusually distended cerebral vessels in the same fish. Anterior is tothe left. (M-R) Hemorrhages are associated with the fragmentation of theunderlying vasculature. (M and P) Representative bright-fieldphotomicrographs of Tg (fli1:EGFP);(gata-1:DsRed) embryos incubated inDMSO or 0.5 mg/L atorvastatin at 2 hpf and imaged at 48-52 hpf. Theasterisk denotes the hemorrhage and the black dotted area shows thefield of interest. Anterior is to the left and dorsal to the top. (N-R)Representative composite confocal Z-stack projections of the blackdotted area in the same Tg(fli1:EGFP);(gata-1:DsRed) embryos. The whiteasterisk denotes DsRed-positive erythrocytes and the white arrows showregions where vascular disintegration is observed.

Measuring the Expression of Cellular Junction Proteins

qRT-PCR was used to assess relative expression of selected genes (anaverage of two biological trial and three technical replicates and eachtrial is a pool of 60 larvae/treatment), using β-actin as thehousekeeping gene control. 3 dpf, Tg(Flk:GFP; Gata:dsRed) zebrafishlarvae were used for RNA extraction and cDNA synthesis. Atorvastatin anddrug treatment were performed as previously mentioned in drug screening.Total RNA was extracted from these larvae (a pool of 50-60larvae/treatment) using the RNeasy extraction kit (Qiagen, Mississauga,ON, CAN) and treated with DNase. The concentration of total RNA wasdetermined spectrophotometrically at 260/280 nm using a NanoDrop™spectrophotometer. First-strand cDNA was synthesized from 1 μg of totalRNA using random hexamer primers.

PCR Conditions:

The genes of interest and the primer pairs used are shown in Table 2. Ineach case, forward primer is shown on the top and reverse primer on thebottom.

TABLE 2 Primer sequences for selected genes to perform qPCR. ProteinGene Primer Sequences SEQ ID name name (5′-3′) NO: VE- Cdh5ACGATGTCTCCATCCTGTCT SEQ ID Cadherin NO: 8 TAGTGATTCGGTTCCCTCAT SEQ IDNO: 9 CCM1 Ccm1 TCACGCTATTCCTGCTCTGT SEQ ID NO: 10 ACTGCAGATCTGAGCCGTACSEQ ID NO: 11 CCM2 Ccm2 GGACAGCCAGCATTTTGAGA SEQ ID NO: 12GTCTGAAATCATGCGGTCCC SEQ ID NO: 13 CCM3 Ccm3 CATGATTGACAGGCCCGAG SEQ IDNO: 14 TGATTGTCTGCAGGAATCGG SEQ ID NO: 15 Integrin Itgb3TCACTGTGGACTTTGCTTGC SEQ ID β3 NO: 16 CACATTCACAGAACGGACCC SEQ ID NO: 17

Amplification of cDNA was achieved with an initial denaturation at 94°C. for 2 min followed by 40 cycles of denaturation (94° C. for 30 sec),annealing (60° C. for 30 sec) and extension (72° C. for 1 min) followedby a final extension period of 10 min at 72° C. before termination. PCRwas carried out in a 20 μl total volume and included 1×PCR buffer, 1.25mM MgCl₂, 0.25 mM dNTP, 1 U Taq polymerase, 0.5 μmol/L forward, andreverse primers and 1 μl cDNA.

Toxicity Assay

The embryos were collected and distributed into a 96-well plate in 0.5%DMSO, similar to efficacy assays. Drugs were added at 24 hpf from arange of 50 nmol/L to 100 μmol/L. 3 days postfertilization (dpf), larvaewere observed for heart beat, blood flow and cardiac edema; the drugtreated larvae were compared to non-treated samples in 0.5% DMSO. Heartbeat and blood flow were ranked from 3 (normal heart beat or blood flow)to 0 (no heart beat or blood flow). Cardiac edema was ranked from 0(normal heart without edema) to −3 (severe cardiac edema). TC50 is theconcentration of the drug at 50% of maximum toxicity. The ratio ofTC50/EC50 was calculated in each case.

Mouse Model Work

Animals and Animal Husbandry

All mice were housed in individually ventilated microisolator cages atSt.

Michael's Hospital vivarium facility. Rooms were kept at an ambienttemperature of 21° C. and subjected to a 12 hour light/dark cycles.Humidity was kept between 30-50%. All mice had access to autoclaved foodand water ad libitum. Virox was used as disinfectant. EnvironmentalEnrichment was provided for mice in each cage. The Animal Care Committeeat St. Michael's Hospital approved all protocols and procedures in thisstudy.

Lipopolysaccharide (LPS)-Induced Microbleeding Model

An LPS-mediated micro bleeding model was created similar to thatdescribed in Lui et al (Liu S, Grigoryan M M, Vasilevko V et al.Comparative analysis of H&E and Prussian blue staining in a mouse modelof cerebral microbleeds. J Histochem Cytochem 2014; 62:767-773). 9-10week old C57BL/6 mice of both sexes were purchased from Charles River,and randomly assigned to control or treatment groups in equal numbers.LPS from Salmonella enterica (Sigma Aldrich, St Louis, Mo.) wasreconstituted with PBS to a final concentration of 5 mg/ml. Both control(n=16) and drug treatment (n=16) groups received injections of 5 mg/kgLPS at times 0 and 24 hrs. The drug treatment group, further dividedinto high dose (n=4) and low dose (n=12), received intraperitonealinjections of artemether (ARM) (25 mg/kg for Low dose and 100 mg/kg forHigh dose) at time points −72, −48, −24, 0 and 24 hrs of LPS treatment.All mice were sacrificed at 48 hrs after the first LPS injection.

The brains were used for either histological studies or MRI study.

Anti-β3 Integrin Model of Intracerebral Hemorrhage (ICH)

An anti-β3 integrin model of intracerebral hemorrhage (ICH) wasgenerated according to our previously reported methods (Yougbare I, LangS, Yang H et al. Maternal anti-platelet beta3 integrins impairangiogenesis and cause intracranial hemorrhage. J Clin Invest 2015;125:1545-1556). Briefly, serum containing anti-β3 antibodies wasgenerated by immunizing β3−/− female mice with gel-filtered wild typeplatelets via tail-vein injections twice a week. To detect anti-β3antibody, blood was collected from the saphenous vein of immunizedfemale mice and left to clot. Serum was extracted by centrifuging bloodat 9600 g for 5 minutes, incubated with FITC-conjugated anti-mouse IgG,and assayed by flow cytometer (FACSCalibur, BD Biosciences, Mississauga,ON).

To generate β3+/− mice, 6-8 week old β3−/− female mice were crossed withwild type male BALB/c. The resulting pups were randomly assigned toeither control (n=29, without any treatment) or drug treatment group(n=24). To induce ICH in the pups, each mouse was injectedintraperitoneally with either 50 μL of the anti-β3 sera (the Controlgroup) or 50 μL anti-β3 sera with 25 mg/kg ARM (Drug Treatment group) atpostnatal day 2 (P2). All Neonates were sacrificed by decapitation atP3.

Histological Studies on Mouse Brains

All histological studies are done according to our established protocols(D'Abbondanza J A, Ai J, Lass E et al. Robust effects of geneticbackground on responses to subarachnoid hemorrhage in mice. J CerebBlood Flow Metab 2016; 36:1942-19547-13; Sabri M, Kawashima A, Ai J,Macdonald R L. Neuronal and astrocytic apoptosis after subarachnoidhemorrhage: a possible cause for poor prognosis. Brain Res 2008;1238:163-171; Sabri M, Jeon H, Ai J et al. Anterior circulation mousemodel of subarachnoid hemorrhage. Brain Res 2009; 1295:179-185; Sabri M,Ai J, Macdonald R L. Dissociation of vasospasm and secondary effects ofexperimental subarachnoid hemorrhage by clazosentan. Stroke 2011;42:1454-1460; Sabri M, Ai J, Marsden P A, Macdonald R L. Simvastatinre-couples dysfunctional endothelial nitric oxide synthase inexperimental subarachnoid hemorrhage. PLoS One 2011; 6:e17062; Sabri M,Ai J, Lakovic K, D'Abbondanza J, Ilodigwe D, Macdonald R L. Mechanismsof microthrombi formation after experimental subarachnoid hemorrhage.Neuroscience 2012; 224:26-37; Sabri M, Ai J, Lass E, D'Abbondanza J,Macdonald R L. Genetic elimination of eNOS reduces secondarycomplications of experimental subarachnoid hemorrhage. J Cereb BloodFlow Metab 2013; 33:1008-1014).

LPS-Induced Microbleeding Model:

All mice in the LPS study were deeply anesthetized with ketamine andxylazine and perfused through the left cardiac ventricle with NaCl,0.9%, followed by 4% paraformaldehyde (PFA) in 1×PBS buffer and 2 mMGadoteridol contrast agent for 24 hours. Each brain was transferred intoa 1×PBS+0.02% sodium azide and 2 mM Gadoteridol contrast agent. Brainswere kept in this immersion solution for 14 days before imaging toensure proper contrast diffusion in the brain for magnetic resonanceimaging (MRI) scan. For non-MRI scan brains, after gross examination,brains were fixed with 4% paraformaldehyde (PFA) in 1×PBS buffer for 24hours and then transferred into a 1×PBS+0.02% sodium azide for storagebefore processing for histology. For histology, brains were cut in amouse brain matrix (Zivic Instruments, Pittsburgh, Pa.). Three (3)coronal cuts were made at −6 mm from bregma, middle line of cerebellum),then 4 mm anterior (−2 mm from bregma) and then 3 mm anterior to thesecond cut (+1 from bregma). Blocks were embedded in paraffin and 7 μmsections cut using a microtome.

Anti-β3 ICH Model:

For a subset of mice (n=16) intended for MRI, the whole head was severedfrom the neck and was immediately fixed with 4% paraformaldehyde (PFA)in 1×PBS buffer and 2 mM Gadoteridol contrast agent for 24 hours. Eachbrain was transferred into a 1×PBS+0.02% sodium azide and 2 mMGadoteridol contrast agent. Heads were kept in this immersion solutionfor 14 days before imaging to ensure proper contrast diffusion in thebrain. For non-MRI brains, after gross examination, brains were fixedwith 4% paraformaldehyde (PFA) in 1×PBS buffer for 24 hours and thentransferred into a 1×PBS+0.02% sodium azide for storage before processedfor histology.

Hematoxylin and Eosin Staining

Brain blocks were processed and embedded in paraffin. Seven micronsections were cut using a microtome. Sections were deparaffinized inxylene and rehydrated through a decreasing gradient of ethanolsolutions. Slides were stained with hematoxylin and eosin, coverslippedwith xylene-based mounting medium (Permount, Sigma Chemical Company, St.Louis, Mo.) and viewed under a light microscope.

Fluoro-Jade Staining

Fluoro-jade B (Histo-Chem Inc., Jefferson, Ark.) was used to assessneuronal degeneration. Brain sections were deparaffinized andrehydrated. Following incubation with deionized water, the slides wereincubated in 0.06% potassium permanganate (Sigma-Aldrich) for 15minutes. Slides were then rinsed in deionized water and immersed for 30minutes in 0.001% Fluoro-jade B working solution (0.1% acetic acid).Slides were washed and dried (60° C.) for 15 minutes, then cleared inxylene and coverslipped with a non-aqueous, low fluorescence, styrenebased mounting media (DPX, Sigma-Aldrich). Slides were viewed under afluorescent light microscope (Olympus BX50, Olympus, Richmond Hill, ON,Canada) and images were taken using constant parameters (exposure timeand contrast values).

Gross Examination

For the integrin ICH model, brains were taken out of the skull, cut atthe mid-coronal position and assessed in a binary manner for whether ornot there was any evidence of ICH. Brains were immediately fixedfollowing assessment. For the LPS model, brains were extracted afterperfusion fixation, and images were taken for the whole brain to examinethe appearance of microbleeding spots.

Contrast Enhanced Magnetic Resonance Imaging

Brains were scanned using 7T Burker Mill with 16-channel solenoid coils.Pulse sequence utilized was a FLASH T2* gradient echo (GRE) sequencewith the following parameters: TR=30.2 ms and TE=12 ms.matrix=250×200×200. FOV=FOV=2.5×2.0×2.0 gcrush=6 tcrush=0.002. FA was11°. (Liu S, Grigoryan M M, Vasilevko V et al. Comparative analysis ofH&E and Prussian blue staining in a mouse model of cerebral microbleeds.J Histochem Cytochem 2014; 62:767-773). Voxel size was 100*100*100Following the reconstruction of images and applying image distortioncorrection algorithms, all brains were processed and analyzed for totalvolume of brain and total volume of hemorrhage. Quantification was doneusing percentage of bleeding (normalized to each brain size).Experimental blinding was done to ensure unbiased work at all levels ofpreparation and analysis. First, samples were prepared and coded notknowing which group they belong to. Secondly, a separate technicianblinded to groups scanned the brains. Lastly, quantification was done ina blinded fashion. All quantifications and 3D reconstructions wereperformed using a combination of Display and Amira processing software.

Spectrophotometer Analysis of Hemoglobin Concentration

Drabkin's reagent (Sigma Aldrich) was used for calorimetricquantification of hemoglobin concentration at 540 nm. C57BL/6 mice wererandomly assigned to three groups (each n=4): Control, Low-dose ARM, andHigh-dose ARM. For ARM groups, three days of 25 mg/kg/day and 100mg/kg/day ARM were administered for low and high dose, respectively.Blood from saphenous vein were collected at day 4 and tested forhemoglobin concentration using UV 3600 Shimadzu spectrophotometer. Astandard curve was generated using a known standard solution ofcyanmethemoglobin, and blood concentrations of Hb was compared to thestandard curve.

Data Analysis and Statistics

A-priori power analysis was done to estimate the number of samples ineach group for a two-tailed, unpaired two-sample t-test with a power of0.8 and a of 0.05 to detect a 1 standard deviation difference inbleeding volume. P values were determined by unpaired, two-tailed t-testwith Welch correction, analysis of variance (ANOVA). All bar graphs andDose-Response curves are expressed as mean±SEM or SD.

Chemicals

For all mouse model work, artemether (80 mg·ml-1) was obtained fromDafra Pharma and diluted 1:15 in fractionated coconut oil, and wasadministered intraperitoneally. For zebrafish work, artesunate werepurchased from Guilin Pharmaceutical (Guangxi, China), together withartemether from Dafra are named GMP drugs. Both ART compounds were alsopurchased from Sigma Aldrich (Sigma) for comparison studies with GMPdrugs.

Example 1. Zebrafish Screen to Identify Lead Compounds

Several models of ICH/BMH in zebrafish have been used, includingstatins, bbh^(m292) and rhd^(mi149) mutants and MOs to reduce expressionof pak2a, βPix, Rap1b and cdh5. In addition, low doses of LPS weredetermined to induce ICH in zebrafish, consistent with the mouse BMHmodel. LPS destabilizes the vasculature and causes vascular leakagethroughout the fish, including in the brain.

FIG. 1 shows the results of experiments conducted using anatorvastatin-induced intracerebral hemorrhage (ICH) model in zebrafishfor chemical screening. Panel (A) is a schematic diagram showing themolecular pathway where statins act. Panels (B)-(G): ICH was induced byapplication of 1 μmol/atorvastatin at 2 hours post fertilization ofembryos from adult wild type or Tg (flk-1: eGFP) and Tg (gata-1:DsRed)zebrafish, and arrayed into 96-well plates that contained the drugcompounds. ICH phenotype rescue was measured. No extravasation of redblood cells was observed in vehicle DMSO treated control embryos (panelsB, D and F). Atorvastatin treated embryos show hemorrhage in the brain(≈80% panels C and G), and increased junction between endothelial cells(panel E as compared to panel D). Panel H is a schematic showing thescheme of the screening process. Panels I to L show EC50 experiments forfour compounds from the ART family, two of which were identified fromthe NCC library. Data is expressed as mean±SEM from 3-4 experiments.ARM, artemether; DHA, dihydro-artemisinin; ARS, artemisinin; ART,artesunate.

Screening of NCC libraries. The National Institutes of Health (NIH)Clinical Collections 1 and 2 consist of 727 compounds including manyFood and Drug Administration-approved drugs for drug repurposing(www.nihclinicalcollection.com). These compounds are mostly drugs thathave been in phase 1 to 3 clinical trials and are not represented onother arrayed collections. They have favorable properties such aspurity, solubility and commercial availability. Many have known safetyprofiles.

After optimizing the brain hemorrhage model with 1 μmol/L atorvastatin,727 compounds in NIH compound libraries 1 and 2(http://nihsmr.evotec.com/evotec/sets/ncc) were screened using theconditions described (96 well plates with 7 embryos per well, andatorvastatin, 1 μmol/L). Six active compounds from four families (twodihydropyridine calcium channel blockers (benidipine and lacidipine),ethynylestradiol, triptolide, two anti-malaria drugs (artesunate andartemether)) were identified independently from the libraries. Chemicalstructure and properties of these six active compounds plus two of thederivatives of ART family compounds are summarized in Table 3 and FIGS.1 and 2.

FIG. 2 shows inhibition of brain hemorrhage induced by E 1 μmol/Latorvastatin in zebrafish by four active compounds identified from NCClibraries, where EC50 is the concentration of the drug at 50% ofefficacy. Data is expressed as mean±SEM from 3 to 4 experiments. Datawas normalized to that of vehicle-treated controls, and fitted withsigmoidal fit with variable slope in GraphPad Prism 4 software. B isbenidipine; E is ethynylestradiol; L is lacidipine, and T is triptolide.

TABLE 3 Active compounds identified from NIH clinical collections(NGP-104 library). The ATV model was induced by 1 μmol/L atorvastatin.One + sign represents 20% inhibition on 1 μmol/L ATV or β-Pix MO-inducedhemorrhage. EC₅₀ (in Property nmol/L) on Efficacy on Efficacy on oratorfvastatin Name of Chemical atorvastatin β-Pix MO Clinical modelCompound Structure Hemorrhage Hemorrhage Application (μmol/L)Artemisinin

+++++ +++++ Anti- malaria  95 Dihydroartemisinin

+++++ +++++ Anti- malaria  67 Artemether (NGP- 104-6-F5)

+++++ +++++ Anti- malaria  64 Artesunate (NGP- 104-2-E7)

+++++ +++++ Anti- malaria 211 Benidipine (NGP- 104-30B7)

++++ ++++ Hyper- tension Lacidipine (NGP- 104-6-C2)

++++ Not tested Hyper- tension Ethynylestadiol (NGP-104-1- E10)

+++ Not tested Contra- ceptive Triptolide (NGP- 104-3-G7)

++++ No effect Not used in clinic; anti- cancer, immuno- suppressive andanti- inflammatory

Three of the four ART compounds showed high potency with EC50 less than100 nmol/L. Due to the moderate potency of the other four compounds(EC50 ranging 191 to 290 nmol/L), we did not investigate them further.

Example 2. Studies on Mechanisms of Action of ART Compounds in Zebrafish

Clinical studies have disclosed a link between cholesterol—lowering3-hydroxy-methylglutaryl-coenzyme A reductase (HMGCR) inhibitors(statins) and increased risk of ICH. The HMGCR pathway is connected tocomponents of the Rho guanosine triphosphatase (GTPase) signalingpathway by prenylation of Cdc42/Rac (FIG. 3). Many proteins in thispathway are responsible for vascular stability. We hypothesized thatsome ICH/BMB are secondary to vascular instability that is mediated byimpaired protein prenylation; and that any defect induced in theproteins (such as mutation or changes in expression) might causehemorrhage. To address the pathways and proteins that are involved andto better understand the mechanism by which a drug rescues thehemorrhage, we decided to induce hemorrhage by genetic modification andto test whether it could be rescued by the ART drugs.

ART Compounds Rescued Bbh Genetic Model of Brain Hemorrhage.

A specific zebrafish line with a gene mutation called bubblehead (bbh)was used. This line has spontaneous ICH. Bubblehead phenotype is causedby a mutation in βPix. Adult homozygous zebrafish were viable andfertile. Bubblehead embryos develop ICH and brain edema 36 to 52 hourspostfertilization (hpf) (Liu J, Zeng L, Kennedy R M, Gruenig N M, ChildsS J. betaPix plays a dual role in cerebral vascular stability andangiogenesis, and interacts with integrin alphavbeta8. Dev Biol 2012;363:95-105; Liu J, Fraser S D, Faloon P W et al. A betaPix Pak2asignaling pathway regulates cerebral vascular stability in zebrafish.Proc Natl Acad Sci USA 2007; 104:13990-13995). More than 85% ofzebrafish larvae display an ICH phenotype. Interestingly, we found thattreating with the ART drugs could completely rescue the hemorrhage inbbh mutants. FIG. 4 shows results from drug efficacy assays in the bbhmodel for two compounds, artesunate (ART), and artemether (ARM). Table 4shows EC50 values measured for the various drugs.

TABLE 4 Comparison of efficacy (EC50 values) of different drugs torescue brain hemorrhage in statin and bbh models. For the statin model,n = 15-20 larvae per condition; the experiment was performed three timesper compound. For the bbh mutant model, n = 15-20 larvae per condition;the experiment was performed 1-3 times per compound. bbh Mutant ModelDrug Statin Model (nmol/L) (nmol/L) ART (GMP) 182.2 126.9 ART (Sigma)105.0 140.3 ARM (Sigma) 24.7 37.5 ARS (Sigma) 81.3 176.6 DHA (Sigma)80.8 107.1

The obtained EC50 values of the ART drugs from the bbh mutant model andfrom the atorvastatin-induced ICH model were comparable. This confirmsthe validity of the statin model which was used for initial screening(Table 3).

ART Compounds Rescued ICH Induced by Gene Knockdown of Key Proteins inthe HMBCR/Rho Kinase Pathway

Besides using bbh, the other method to induce ICH in zebrafish isgenetic gene knockdown. We used specific morpholinos to knock down somekey genes in both HMGCR and Rho guanosine triphosphatase (GTPase)signaling pathways (FIG. 3). The morpholinos for the following geneswere used:

1) Pak2a: p21 protein (Cdc42/Rac)-activated kinase 2a regulates activityof Rho GTPases, Rac and Cdc42, and may be involved in a complex withβPix.

2) βPix: Pak-interacting exchange factor β facilitates conversion ofGDP-Rho GTPases (Rac and Cdc42) to GTP-RhoGTPase.

3) HMGCR: 3-hydroxy-3-methylglutaryl-coenzyme A reductase catalyzesconversion of HMG-Co A to mevalonate.

4) VE-Cadherin: Vascular Endothelial Cadherin is a transmembrane proteinthat connects the intracellular cytoskeleton to the extracellularmatrix.

5) Rap1b: Ras GTPase effector protein facilitates recruiting of CCMproteins to the cell membrane.

6) GGTase 1: geranylgeranyltransferase 1 post-translationally modifiesRac and Cdc42 by adding a mevalonate-derived GGPP which is required toactivate these GTPases.

FIG. 14 (A-B) shows that artesunate dose-dependently rescues hemorrhagephenotype induced by morpholinos targeting membrane stability of brainvessels in zebrafish. (A) Schematic diagram showing the target sites ofthe three morpholinos studied. (B) Artesunate dose-dependently rescuesall three morpholinos-induced brain hemorrhage in zebrafish. (C-D)Artesunate rescues the ICH phenotype underlying the bbh^(m292) mutation.(C) Upper panel, partial exon-intron organization of bPix gene showingthe point mutation effecting splicing of the gene. Lower panel, RT-PCRanalysis of wild-type and bbh^(m292) mutant cDNA with primers flankingexon-14. (D) Upper panel, the phenotypes of bbh^(m292) mutants treatedwith DMSO or artesunate and imaged at 48 hpf. The arrows denote sites ofhemorrhage. Lower panel, percentages of bbh^(m292) embryos with brainhemorrhage rescued by artesunate.

Artesunate dose-dependently reduced ICH/BMH after treatment withpak2a-MO (FIGS. 12 and 14). MO-mediated inhibition of hmgcrb, thezebrafish enzyme inhibited by statins, caused embryos to have ICH/BMHwhich also were prevented by artesunate (FIG. 14).

Finally, a role for the VE-cadherin homologue in zebrafish, cdh5, wasdemonstrated in that MO-knockdown of cdh5 induced ICH in zebrafish (FIG.13). FIG. 13 shows the HMGCR molecular pathway that leads to vascularstability in zebrafish. Panels A & B: Stable EC junctions are maintainedby a Cdc42-dependent and VE-cadherin-mediated cell-cell adhesion. VEcadherins are found on the surfaces of EC cell-cell junctions.VE-cadherins are associated with β- and α-catenins at their cytoplasmicdomains, which connect them to the actin-based cytoskeleton (bluecircles). Cdc42 belongs to the Rho-family of small guanosinetriphosphatases (GTPases), which are the main regulators ofVE-cadherin-based cell-cell adhesion. The functions of hmgcrb, βPix, andpak2a in regulating junctional stability in zebrafish are shown. HNGCRmediated GGPP biosynthesis regulates Cdc42 prenylation. βPix is a GEFthat increases CDC42 affinity for GTP. Pak2 is an effector of Cdc42,which regulates actin filament organization. Panel C shows thatsplice-inducing morpholinos designed against cdh5, the zebrafishortholog of the VE-cadherin gene, induced intracerebral hemorrhage inzebrafish at 36-48 hpf (lateral images are shown).

We found that injection of any of the above morpholinos causeshemorrhage in 3 dpf zebrafish larvae indicating the important rolesthese genes play in vascular stability. Next, optimum amount of eachmorpholino was determined. The goal was to induce an acceptablepercentage of hemorrhage (ideally between 40-80%), without havingtoxicity from morpholino injection. The results are summarized in Table5.

TABLE 5 Optimizing the amount of injected morpholinos, n = 150-250larvae per condition; the experiment was performed at least two times.Morpholino Optimum amount (ng) Hemorrhage βPix - exon6 0.8 72.1% Pak2a -exon8 5.0 43.7% Hmgcrb - splice 2.0 67.9% Cdh5 - exon2 1.0 39.4% 35.0%(Cardiac Edema) Rap1b - exon3 9.0 30.8% (Faint) GGTasel 2.5 11.4%(Faint)

The first three morpholino pairs were good for the efficacy study.Efficacy assays were performed using Aartemether (ARM, Sigma) andartemesunate (ART, GMP). The results showed that defects induced byPak2a, βPix, and HMGCR morpholinos could induce hemorrhage inindependent experiments. Treating these morphants with the drugs rescuedthe ICH phenotype. FIG. 5 shows an example of a drug efficacy study onhmgcrb morphants using artesunate (ART), and artemether (ARM); n=15-20larvae per condition; the experiment was performed two times percompound. EC50 values calculated for the first three aforementionedmorphants are shown in Table 6.

TABLE 6 Efficacy comparison (EC50) of ARM (Sigma) and ART (GMP) torescue the hemorrhage induced by different morpholinos; n = 150-250larvae per condition, and the experiment was performed at least twotimes. Morpholino ARM (Sigma) (nmol/L) ART (GMP) (nmol/L) βPix - exon6 51.6 ± 11.7  95.6 ± 27.9 Pak2a - exon8 39.1 ± 8.0 169.2 ± 39.5 Hmgcrb -splice 62.5 ± 8.3 166.7 ± 9.3  Cdh5 - exon2 Rescue effect Rescue effect

Suppression of VE-cadherin induces hemorrhage. However, cardiac edemawas observed in some morphants. The efficacy assays were performed usingARM and ART, and in both cases rescue was observed.

Consistent with what we found in the atorvastatin-induced ICH model andbbh mutant model, ARM showed the highest efficacy to rescue thehemorrhage in morphants (Table 4 and Table 6).

Studies on Toxicity of ART Compounds in Zebrafish

Toxicity assays were performed to measure TC50 values considering threeparameters: heartbeat, blood flow and cardiac edema As an example, FIG.7 shows the results of toxicity assays for ART (GMP). Heart beat andblood flow were ranked from 3 (normal heart beat or blood flow) to 0 (noheart beat or blood flow). Cardiac edema was ranked from 0 (normal heartwithout edema) to −3 (severe cardiac edema). TC50 is the concentrationof the drug at 50% of maximum toxicity. Data is expressed as mean±SEMfrom 3 experiments. TC50 values for all drugs as well as the TC50/EC50ratio are summarized in Table 7.

TABLE 7 Comparison of toxicity (TC50 values) of different drugs.TC50/EC50 ratio is in parenthesis; n = 15-20 larvae per condition, andthe experiment was performed three times per compound. TC50 TC50 TC50(Heart beat) (Blood flow) (Edema) Drug Company [nmol/L] [nmol/L][nmol/L] Artemisinin (ARS) Sigma 8501 1058   34134  (104.5) (13)   (419)Artesunate (ART) Sigma 8884 741.5 5367 (84.6)  (7.1)    (51.1)Artemether (ARM) Sigma 2328 975.3 19564  (94.3)  (39.5)    (87.1)Dihydroartemisinin Sigma  5.011e+0.11 890     748.5 (DHA)  (6.2e+009) (11.0)     (9..2) Artemisinin (ARS) Sequoia Rsearch 1.217e+010 5340  6347 (UK)    4.6e+007)  (20.4)    24.2) Artesunate (ART) Sequoia 95391036     937.8 Research (UK) (59.5)  (6.5)    (5.9) Artemether (ARM)Sequoia 1.084e+013 746.3 5.253e+012 Research (UK)  (1.3e+011)  (9.2) (6.5e+010) Dihydroartemisinin Sequoia 1.110e+008 979.5 1021 (DHA)Research (UK)  (8.3e+005)  (7.3)    (7.6) Artesunate (ART)GMP-Artesunate 2525 794.4 3196 (13.9)  (4.4)    (17.5) ZA102 LifeChemicals 376 695.3 24444  (2.1)  (3.8)  (135) ZA113 Life Chemicals 2283572.3 5883 (17.3)  (4.3)    (44.5) ZA123 Life Chemicals 6.491e+007 989.355912   (5.2e+005)  (7.9)   (445.5)

We found ARM to be a safe drug, as its EC50 is much lower than TC50(Table 7) in zebrafish embryos.

3) ART Compounds Upregulate Key Proteins Vital for Vascular Stability

We considered a list of 20 genes that are potentially involved in ICHmechanism, and evaluated the changes in transcription of five of themafter in atorvastatin-induced brain hemorrhage model and treated withART drugs. These genes are: VE-Cadherin (Cdh5), Integrin (Itgb3a) andthree cerebral cavernous malformation genes (ccm1, ccm2 and ccm3).

FIG. 6 shows the changes in gene transcription upon adding statin (ATV)and artemether (ARM) at 500 nmol/L. qRT-PCR analysis was used toevaluate the mRNA level of gene expression of A, VE-Cadherin; B,β3-Integrin, C CCM3, in zebrafish treated with atorvastatin (ATV) and500 nmol/L of Artemether (ARM), n=3. At this concentration, nohemorrhage was observed. Each figure shows the experiment results of50-60 of 3 dpf embryos.

The results showed that in the statin-induced ICH model, upregulation atthe transcription level occurs for Integrin β3 and VE-cadherin upontreatment with ARM. CCM3 showed a decrease after inducing hemorrhagewith atorvastatin. Upon treatment with ARM, the transcription levelreturned to normal in parallel with hemorrhage rescue in zebrafishlarvae.

Example 3. Other Zebrafish Models to Validate Anti-ICH Efficacy ofCompounds Identified from Statin-Derived Embryonic Screens

Experiment 1: LPS-Induced ICH/BMH in Zebrafish Embryos.

The lead compounds will be tested in a LPS model of ICH/BMH to determineif rescue of the ICH/BMH phenotype is a general property of thesecompounds or it is specific to statin-induced ICH/BMH. Preliminary datasuggested that artemether reduced mortality from LPS (FIG. 15). FIG. 15shows that LPS induces brain hemorrhage in developing zebrafish embryoand artemether have protective effects on LPS-induced mortality. Panel Ashows survival curves of developing zebrafish embryos when LPS isdelivered in fish water at 24 hours post fertilization (hpf). Panel Bshows that 1 μmol/L artemether in fish water had a protective effect onfish survival. LPS concentration used was 200 mg/mL. Panel C shows that25 mg/mL LPS treatment of 24 hpf embryos resulted in no mortality but52% of embryos (n=120) had brain hemorrhage. Experiments are ongoing todefine the rescuing effects of artemether on LPS-induced brainhemorrhage. Double transgenic zebrafish (Gata1:DsRed/Flk1:GFP) withgreen fluorescent vessel and red fluorescent red blood cells are used.Arrow points to hemorrhage.

Example 4. Work in Mouse Models of ICH

We employed two models of brain hemorrhage. Our results show that inboth LPS and Integrin models of ICH, ARM effectively prevented orameliorated hemorrhage.

LPS-Induced Microbleeding Mouse Model

ARM (GMP) Reduces Both Surface and Deep Brain Microbleeds Induced by LPS

LPS and its main receptor TLR4 have been extensively studied, and recentliterature characterized a model of brain micro-bleeds that are bothpresent on the surface cortical areas and in the deep lobar areas (LiuS, Grigoryan M M, Vasilevko V et al. Comparative analysis of H&E andPrussian blue staining in a mouse model of cerebral microbleeds. JHistochem Cytochem 2014; 62:767-773; Sumbria R K, Grigoryan M M,Vasilevko V et al. A murine model of inflammation-induced cerebralmicrobleeds. J Neuroinflammation 2016; 13:218). The number of surfacemicro-bleeds of each brain was counted using a stereomicroscope, and anaverage determined for LPS control and LPS+ARM treatment groups. FIG. 8shows that artemether (ARM) rescues LPS-induced brain microbleeds inmice. Panel A shows data from a stereomicroscope count of surfacemicrobleeds in brains from LPS treated mice (n=8) orLPS+artemether-treated mice (n=8). The left panel shows representativeimages from each of the two groups; arrows indicate microbleeds. Theright panel shows a statistical analysis (*P<0.05, two-tailed t-testwith Welch correction); data is expressed as mean±SD. As compared to LPStreated animals, brains from ARM treated mice showed a robust reductionin total surface microbleeds.

To further assess microbleeding inside the brains, we quantified thenumbers of microbleeds in H&E stained brain slides. Panel B shows datafrom quantification of microbleeds on brain slices stained byhematoxylin and eosin. The left panel shows representative images ofstained brain slices with microbleeds from each of the two groups; thearrows indicate microbleeds on the slices; the right panel chart shows astatistical analysis on microbleeds count (**P<0.01, unpaired two-tailedt-test with Welch's correction. Data is expressed as mean±SD, n=8 forboth LPS treated and LPS+ARM treated groups.

Similar to the surface microbleed counts, ARM treatment significantlyreduced the total number of microbleeds inside the mouse brains (FIG.8B).

The Reduction of Total LPS-Induced Microbleeds in Mouse Brains by ARM(GMP) is Verified by MRI

To confirm the result from gross anatomy and histology, we examined thebrains in the subsequent experiments using a Mill with 3D FLASH GREsequence. Total volume of hemorrhage was quantified and percent bleedingwas calculated for each brain. FIG. 9 shows that artemether (ARM)rescues microbleeding induced by lipopolysaccharide (LPS) in mice. PanelA shows representative 3D reconstructed images from T2*−WeightedGradient Echo (GRE) MRI sequence with high resolution detecting, showingmicrobleeds from LPS or LPS+ARM treated mouse brains. Arrows indicatethe microbleeds. Panel B is a bar graph showing the number ofmicrobleedings per brain in a vehicle control group and a group treatedwith artemether (ARM). Quantification of total microbleeds volume wascalculated using semi-automated software (Display), normalized to totalbrain volume, and expressed as total voxel in 10000 counts. Data isexpressed as mean±SD (+P<0.05, two-tailed t-test with Welch correction),n=8 for both LPS treated and LPS+ARM treated groups, 2 for naïvecontrols.

The data confirm that there is a significant reduction of bleeding(about ⅔ reduction) in the ARM treated group in comparison to the modelcontrol group (FIG. 9).

LPS Did not Induce Significant Neuronal Cell Death or HemosiderinDeposition

We did Fluoro-jade C and Perl's staining to detect neuronal degenerationand hemosiderin deposition, respectively. The results of both of theseassays were negative for both control and treatment groups, suggestingthat the observed micro-bleeds induced by LPS are acute and that themicrobleeds did not cause neuronal cell death, at least in the timescale we tested on this model.

Integrin ICH Mouse Model

ARM (GMP) Reduces the Incidence Rate of ICH

Previous studies suggested that by forming a heterodimer with the αVsubunit of integrin, β3 integrin plays a role in proliferatingendothelial cells, specifically during angiogenesis (Yougbare I, Lang S,Yang H et al. Maternal anti-platelet beta3 integrins impair angiogenesisand cause intracranial hemorrhage. J Clin Invest 2015; 125:1545-1556).It has already been shown that using antibodies especially during thedevelopmental stage creates vascular instability and improperangiogenesis and hence rapid ICH development. Id.

We employed two end points to examine the treatment effect of ARM in theanti-β3 integrin model of intracerebral hemorrhage. FIG. 10 shows thatartemether (ARM) reduces ICH in an anti-β3 integrin mouse model ofintracerebral hemorrhage. Panel A shows representative raw T2*-WeightedGradient Echo (GRE) MRI images of brains of mice injected with anti-β3integrin serum at post-natal day 2 alone (left) or treated with ARM(right). Panel B shows paraffin-embedded blocks of coronally-cut wholebrains from anti-β3 serum injected mice without (left) or with (right)ARM treatment, respectively. Panel C shows quantification of frequencyof intracerebral hemorrhage in mice injected with anti-β3 integrin serumalone or with ARM treatment. 77% of neonates showed ICH in the ICH modelcontrol group. In comparison, ARM reduced ICH incidence to 47%.Data isexpressed as mean±SD (**P<0.01, two-tailed t-test with Welchcorrection), n=29 and 24 for anti-β3 integrin serum injected micewithout or with ARM treatment, respectively.

ARM (GMP) Reduces the Total Volume of ICH Verified by MRI

Preliminary data shows that ARM reduced total volume of ICH as comparedto controls. (data not shown).

To assess possible anemia effect from ARM treatment as some previousstudies speculated, blood samples were tested for hemoglobinconcentration after ARM treatment. Blood hemoglobin concentration wasassessed using Drabkins' method. Spectrophotometer data was compared toa standard curve from standard cyanmethemoglobin concentrations. Thecontrol group received no drug. The Treatment Dose group received 3 daysinjection of low dose ARM (25 mg/kg), 4× Treatment Dose group received 3days injection of high dose ARM (100 mg/kg).

FIG. 11 shows that ARM treatment for 3 days did not cause anemia inmice. It is a plot of blood hemoglobin (g/dl) (y-axis) for controls, andfor mice treated with artemether (ARM) (Treatment Dose, and 4× TreatmentDose). Bloods were tested for hemoglobin concentration after ARMtreatment. Blood hemoglobin concentration was assessed using Drabkins'method. Spectrophotometer data was compared to a standard curve fromstandard cyanmethemoglobin concentrations. The control group received nodrug. The Treatment Dose group received 3 days injection of low dose ARM(25 mg/kg); 4× Treatment Dose group received 3 days injection of highdose ARM (100 mg/kg). Data is expressed as mean±SD (nsP>0.05, one-wayANOVA, n=4). We did not find any statistical difference between thegroups (FIG. 11).

FIG. 16 shows that statin exacerbates LPS-induced intracerebralhemorrhage in mice. (A) Atorvastatin (50 mg/kg) treatment in addition toLPS (5 mg/kg), resulted in 100% mortality 24 hours after the treatments,while LPS treatment alone only result in 25% mortality at the same timeexamined, and statin alone did not cause any mortality (n=5). (B)Atorvastatin treatment significantly increased the number of largehemorrhages caused by LPS. While the present invention has beendescribed with reference to the specific embodiments thereof it shouldbe understood by those skilled in the art that various changes may bemade and equivalents may be substituted without departing from the truespirit and scope of the invention. In addition, many modifications maybe made to adopt a particular situation, material, composition ofmatter, process, process step or steps, to the objective spirit andscope of the present invention. All such modifications are intended tobe within the scope of the claims appended hereto.

What is claimed is:
 1. A method for reducing incidence of vascularleakage in the brain comprising administering to a subject in needthereof a pharmaceutical composition containing a small moleculetherapeutic compound selected from the group consisting of artemisininor a derivative of artemisinin, a therapeutic amount of which iseffective to reduce incidence of bleeding in the brain, wherein thebrain vascular leakage is an induced brain microhemorrhage or aspontaneous intracerebral hemorrhage.
 2. The method according to claim1, wherein the derivative of artemisinin is dihydroartemisinin,artemether, or artesunate.
 3. The method according to claim 1, whereinthe small molecule therapeutic compound is selected from the groupconsisting of benidipine, lacidipine, ethynylestradiol or triptolide. 4.The method according to claim 1, wherein the vascular leakage is aninduced vascular leakage, an induced brain hemorrhage or a brainmicrohemorrhage.
 5. The method according to claim 1, wherein thevascular leakage is induced by a statin, by a lipopolysaccharide, orboth.
 6. The method according to claim 4, wherein the statin isatorvastatin.
 7. The method according to claim 1, wherein the vascularleakage is a spontaneous intracerebral hemorrhage.
 8. The methodaccording to claim 1, wherein the brain vascular leakage isaging-related or related to a neural degenerative disease.
 9. The methodaccording to claim 6, wherein the spontaneous intracerebral hemorrhageoccurs in association with a mutation of one or more genes selected frombeta-pix, Pak2a, cdh5, ccm1, ccm2, ccm3, and Rap1b.
 10. The methodaccording to claim 1, wherein the vascular leakage includes a brainmicrohemorrhage.
 11. The method according to claim 8, wherein the brainmicrohemorrhage occurs in association with administration of a statin.12. The method according to claim 1, wherein the vascular leakagecomprises a brain vascular malformation.
 13. The method according toclaim 10, wherein the brain vascular malformation is a cerebralcavernous malformation.
 14. The method according to claim 1, wherein thebrain hemorrhage or brain microhemorrhage is induced by dysfunction ofβ3 integrin signaling.
 15. The method according to claim 14, wherein thedysfunction of β3 integrin signaling is associated with a disease stateselected from the group consisting of a spontaneous intracerebralhemorrhage, an aging-related vascular leakage, an aging-relatedhemorrhage, an aging-related microhemorrhage, a vascular leakage from aneural degenerative disease, a hemorrhage from a neural degenerativedisease, a microhemorrhage from a neural degenerative disease, a brainvascular malformation, or a cerebral cavernous malformation.
 16. Amethod for screening compounds effective to reduce incidence of avascular leakage in brain comprising (i) administering to a zebrafishembryo a pharmaceutical composition containing a statin or LPS; (ii)inducing in the zebrafish embryo a vascular leakage or a brainhemorrhage; and (iii) administering to the zebrafish embryo a compoundeffective to reduce incidence of the vascular leakage or brainhemorrhage.